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Sample records for factor xa inhibitor

  1. Phenyltriazolinones as potent factor Xa inhibitors.

    PubMed

    Quan, Mimi L; Pinto, Donald J P; Rossi, Karen A; Sheriff, Steven; Alexander, Richard S; Amparo, Eugene; Kish, Kevin; Knabb, Robert M; Luettgen, Joseph M; Morin, Paul; Smallwood, Angela; Woerner, Francis J; Wexler, Ruth R

    2010-02-15

    We have discovered that phenyltriazolinone is a novel and potent P1 moiety for coagulation factor Xa. X-ray structures of the inhibitors with a phenyltriazolinone in the P1 position revealed that the side chain of Asp189 has reoriented resulting in a novel S1 binding pocket which is larger in size to accommodate the phenyltriazolinone P1 substrate.

  2. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa.

    PubMed

    Lu, Genmin; DeGuzman, Francis R; Hollenbach, Stanley J; Karbarz, Mark J; Abe, Keith; Lee, Gail; Luan, Peng; Hutchaleelaha, Athiwat; Inagaki, Mayuko; Conley, Pamela B; Phillips, David R; Sinha, Uma

    2013-04-01

    Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

  3. Edoxaban: a new oral direct factor xa inhibitor.

    PubMed

    Camm, A John; Bounameaux, Henri

    2011-08-20

    Edoxaban is an oral direct factor Xa inhibitor that is currently undergoing investigation in phase III clinical trials for the prevention of stroke in patients with atrial fibrillation (AF) and for the prevention and treatment of venous thromboembolic events (VTE). Factor Xa is an attractive target for anticoagulant treatment, as it is the primary and rate-limiting source of amplification in the coagulation cascade. Edoxaban is a competitive inhibitor of factor Xa and has >10 000-fold greater selectivity for factor Xa relative to thrombin. In phase I clinical trials, the anticoagulant effects of edoxaban included dose-dependent increases in activated partial thromboplastin time and prothrombin time following single edoxaban doses of 10-150 mg and after multiple ascending doses (60 mg twice daily, 90 mg daily and 120 mg daily). The anticoagulant effects of edoxaban were rapid in onset (time to peak plasma concentration 1-2 hours) and sustained for up to 24 hours. Prolongation of bleeding time in 8% of subjects was >9.5 minutes (none of which appeared to be clinically significant) 2 hours after initial dosing, and was independent of edoxaban dose, formulation or dietary state. In general, plasma edoxaban concentrations were linearly correlated with coagulation parameters. Phase II clinical trials in patients with AF and VTE suggest that the edoxaban 30 mg once-daily and 60 mg once-daily regimens had a similar or better safety profile compared with dose-adjusted warfarin (international normalized ratio 2.0-3.0) in terms of bleeding events, and that edoxaban was not associated with hepatotoxicity. In addition, edoxaban was associated with statistically significant dose-dependent reductions in VTE after orthopaedic surgery compared with placebo or dalteparin sodium. Further clinical investigation of the efficacy and safety of once-daily edoxaban is being conducted in phase III clinical trials in comparison with warfarin in patients with AF in the phase III

  4. Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

    PubMed

    Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

    2003-07-01

    The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

  5. Role of Factor Xa Inhibitors in Cancer-Associated Thrombosis: Any New Data?

    PubMed Central

    Zalpour, Ali; Kroll, Michael H.; Afshar-Kharghan, Vahid; Yusuf, Syed Wamique; Escalante, Carmen

    2011-01-01

    The association between cancer and venous thromboembolism (VTE) has been well documented in the literature. Prevention and treatment of VTE in cancer patients is imperative. Typically, the mainstay regimen for VTE prevention and treatment has been anticoagulation therapy, unless contraindicated. This therapy consists of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), factor Xa inhibitor, or vitamin K antagonist (VKA). Current guidelines recommend LMWH over VKA for the treatment of VTE in cancer patients. Factor-specific anticoagulants have been proven safe and effective, and recently factor Xa inhibitors have emerged as a treatment alternative to heparins and VKA. Currently, three factor Xa inhibitors have been identified: fondaparinux (the only one approved so far by the US Food and Drug Administration), idraparinux (in clinical trials), and idrabiotaparinux (in clinical trials). This paper will examine the role of these agents, focusing on fondaparinux, for the prevention and treatment of VTE in cancer patients. PMID:22013445

  6. Kinetic characterization of the protein Z-dependent protease inhibitor reaction with blood coagulation factor Xa.

    PubMed

    Huang, Xin; Swanson, Richard; Broze, George J; Olson, Steven T

    2008-10-31

    Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (K(M) 53+/-5 nM) followed by conversion to a stable ZPI-FXa complex (k(lim) 1.2+/-0.1 s(-1)). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.

  7. Pharmacophore identification, in silico screening, and virtual library design for inhibitors of the human factor Xa.

    PubMed

    Krovat, Eva M; Frühwirth, Karin H; Langer, Thierry

    2005-01-01

    Factor Xa inhibitors are innovative anticoagulant agents that provide a better safety/efficacy profile compared to other anticoagulative drugs. A chemical feature-based modeling approach was applied to identify crucial pharmacophore patterns from 3D crystal structures of inhibitors bound to human factor Xa (Pdb entries 1fjs, 1kns, 1eqz) using the software LIGANDSCOUT and CATALYST. The complex structures were selected regarding the criteria of high inhibitory potency (i.e. all ligands show K(i) values against factor Xa in the subnanomolar range) and good resolution (i.e. at least 2.2 A) in order to generate selective and high quality pharmacophore models. The resulting chemical-feature based hypotheses were used for virtual screening of commercial molecular databases such as the WDI database. Furthermore, a ligand-based molecular modeling approach was performed to obtain common-feature hypotheses that represent the relevant chemical interactions between 10 bioactive factor Xa inhibitors and the protein, respectively. In a next step a virtual combinatorial library was designed in order to generate new compounds with similar chemical and spatial properties as known inhibitors. The software tool ILIB DIVERSE was used for this procedure in order to provide new scaffolds of this group of anticoagulants. Finally we present the combination of these two techniques, hence virtual screening was performed with selective pharmacophore models in a focused virtual combinatorial database. De novo derived molecular scaffolds that were able to adequately satisfy the pharmacophore criteria are revealed and are promising templates for candidates for further development.

  8. Chromogenic laboratory assays to measure the factor Xa-inhibiting properties of apixaban--an oral, direct and selective factor Xa inhibitor.

    PubMed

    Becker, Richard C; Yang, Hongqiu; Barrett, Yuchen; Mohan, Puneet; Wang, Jessie; Wallentin, Lars; Alexander, John H

    2011-08-01

    An ability to readily determine an anticoagulant effect with an emerging class of direct, active site, oral factor Xa inhibitors is viewed by the medical community as attractive and by some as an absolute requirement for their use in clinical practice. We performed a pharmacokinetic and pharmacodynamic substudy in APPRAISE-1-a study of apixaban in patients with acute coronary syndrome(ACS). A total of 1691 patients had blood sampled for apixaban plasma concentrations using mass spectrometry/high performance liquid chromatography and anti-Xa activity using a chromogenic assay employing either low molecular weight heparin or apixaban as reference standards. Anti-Xa activity, determined by either anti-Xa-LMWH (r = 0.9671; P < 0.0001) or anti-Xa-apixaban (r = 0.9669; P < 0.0001) correlated strongly and in a linear fashion with apixaban plasma concentrations. The correlations for each method were equally strong at low (<100 ng/ml) (r = 0.86, P < 0.0001; r = 0.85, P < 0.0001), intermediate(100-200 ng/ml) (r = 0.73, P < 0.0001; r = 0.69, P < 0.0001) and high (>200 ng/ml) (r = 0.91, P < 0.0001; r = 0.91, P < 0.0001) plasma concentrations of apixaban, respectively. Our pharmacokinetic and pharmacodynamic substudy suggests that an apixaban-mediated anticoagulant effect can be detected even at very low plasma concentrations using a standard laboratory chromogenic anti-Xa assay with either LMWH or apixaban calibrators. While establishing parameters for safety and efficacy will require further investigation, an ability to discern the presence of a drug effect may provide clinically useful information.

  9. New advances in the discovery of thrombin and factor Xa inhibitors.

    PubMed

    Vacca, J P

    2000-08-01

    The search for the ideal anticoagulant has spanned decades and has resulted in several strategies including the clinical use of heparin, low molecular weight heparins, and the vitamin K antagonist warfarin. Over the past five years, many groups have reported preclinical results with direct-acting thrombin inhibitors and several of these are now moving into clinical trials. In addition, many groups have disclosed the discovery of potent, orally bioavailable factor Xa inhibitors. Several of these compounds are now in early clinical trials and the results are forthcoming.

  10. Anticoagulation beyond direct thrombin and factor Xa inhibitors: indications for targeting the intrinsic pathway?

    PubMed

    van Montfoort, Maurits L; Meijers, Joost C M

    2013-08-01

    Antithrombotic drugs like vitamin K antagonists and heparin have been the gold standard for the treatment and prevention of thromboembolic disease for many years. Unfortunately, there are several disadvantages of these antithrombotic drugs: they are accompanied by serious bleeding problems, it is necessary to monitor the therapeutic window, and there are various interactions with food and other drugs. This has led to the development of new oral anticoagulants, specifically inhibiting either thrombin or factor Xa. In terms of effectiveness, these drugs are comparable to the currently available anticoagulants; however, they are still associated with issues such as bleeding, reversal of the drug and complicated laboratory monitoring. Vitamin K antagonists, heparin, direct thrombin and factor Xa inhibitors have in common that they target key proteins of the haemostatic system. In an attempt to overcome these difficulties we investigated whether the intrinsic coagulation factors (VIII, IX, XI, XII, prekallikrein and high-molecular-weight kininogen) are superior targets for anticoagulation. We analysed epidemiological data concerning thrombosis and bleeding in patients deficient in one of the intrinsic pathway proteins. Furthermore, we discuss several thrombotic models in intrinsic coagulation factor-deficient animals. The combined results suggest that intrinsic coagulation factors could be suitable targets for anticoagulant drugs.

  11. Structure-based drug design of pyrrolidine-1, 2-dicarboxamides as a novel series of orally bioavailable factor Xa inhibitors.

    PubMed

    Van Huis, Chad A; Bigge, Christopher F; Casimiro-Garcia, Agustin; Cody, Wayne L; Dudley, Danette A; Filipski, Kevin J; Heemstra, Ronald J; Kohrt, Jeffrey T; Narasimhan, Lakshmi S; Schaum, Robert P; Zhang, Erli; Bryant, John W; Haarer, Staci; Janiczek, Nancy; Leadley, Robert J; McClanahan, Thomas; Thomas Peterson, J; Welch, Kathleen M; Edmunds, Jeremy J

    2007-06-01

    A novel series of pyrrolidine-1,2-dicarboxamides was discovered as factor Xa inhibitors using structure-based drug design. This series consisted of a neutral 4-chlorophenylurea P1, a biphenylsulfonamide P4 and a D-proline scaffold (1, IC(50) = 18 nM). Optimization of the initial hit resulted in an orally bioavailable, subnanomolar inhibitor of factor Xa (13, IC(50) = 0.38 nM), which was shown to be efficacious in a canine electrolytic model of thrombosis with minimal bleeding.

  12. A NMR and MD study of the active site of factor Xa by selective inhibitors

    NASA Astrophysics Data System (ADS)

    Doan, B. T.; Fraternali, F.; Do, Q. T.; Atkinson, R. A.; Palmas, P.; Sklenar, V.; Wildgoose, P.; Strop, P.; Saudek, V.

    1998-02-01

    The structure of two selective inhibitors obtained by the screening of a vast combinatorial library, Ac-Tyr-Ile-Arg-Ile-NH2 and Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, in the active site of the blood clotting enzyme factor Xa was determined using transferred NOE NMR and simulated annealing (SA) under NMR constraints. The refined structures of the inhibitors were docked in the active site and SA was performed inside the enzyme which has been kept as a rigid charged template. The final structures were optimised by molecular dynamics simulation of the complexes in water. The inhibitors assume a compact, very well defined conformation embedded in the binding site without blocking the catalysis. The model allows to explain the mode of action, affinity and specificity. L'étude structurale d'inhibiteurs du facteur Xa, une enzyme de coagulation, obtenus par chimie combinatoire : Ac-Tyr-Ile-Arg-Ile-NH2, Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, a été réalisée par RMN NOE de transfert et modélisation moléculaire. Les structures ont été calculées sous contraintes RMN : géométrie de distance, recuit simulé et minimisation, affinées par une recherche conformationnelle et recuit de l'inhibiteur placé dans le site actif et optimisées par simulation de dynamique moléculaire du complexe dans l'eau. L'inhibiteur présente une structure compacte positionnée dans le site d'interaction hors d'accès du site catalytique. Ce modèle permet d'expliquer le mode d'action, l'affinité et la spécificité des peptides.

  13. Parenteral administration of factor Xa/IIa inhibitors limits experimental aortic aneurysm and atherosclerosis

    PubMed Central

    Moran, Corey S.; Seto, Sai-Wang; Krishna, Smriti M.; Sharma, Surabhi; Jose, Roby J.; Biros, Erik; Wang, Yutang; Morton, Susan K.; Golledge, Jonathan

    2017-01-01

    Intraluminal thrombus is a consistent feature of human abdominal aortic aneurysm (AAA). Coagulation factor Xa (FXa) catalyses FII to thrombin (FIIa). We examined the effect of FXa/FIIa inhibition on experimental aortic aneurysm in apolipoprotein E-deficient (ApoE−/−) mice infused with angiotensin II (AngII). The concentration of FXa within the supra-renal aorta (SRA) correlated positively with SRA diameter. Parenteral administration of enoxaparin (FXa/IIa inhibitor) and fondaparinux (FXa inhibitor) over 14 days reduced to severity of aortic aneurysm and atherosclerosis in AngII-infused ApoE−/− mice. Enteral administration of the FIIa inhibitor dabigatran had no significant effect. Aortic protease-activated receptor (PAR)-2 expression increased in response to AngII infusion. Fondaparinux reduced SRA levels of FXa, FIIa, PAR-2, matrix metalloproteinase (MMP)2, Smad2/3 phosphorylation, and MOMA-2 positive cells in the mouse model. FXa stimulated Smad2/3 phosphorylation and MMP2 expression in aortic vascular smooth muscle cells (VSMC) in vitro. Expression of MMP2 in FXa-stimulated VSMC was downregulated in the presence of a PAR-2 but not a PAR-1 inhibitor. These findings suggest that FXa/FIIa inhibition limits aortic aneurysm and atherosclerosis severity due to down-regulation of vascular PAR-2-mediated Smad2/3 signalling and MMP2 expression. Inhibition of FXa/FIIa may be a potential therapy for limiting aortic aneurysm. PMID:28220880

  14. Parenteral administration of factor Xa/IIa inhibitors limits experimental aortic aneurysm and atherosclerosis.

    PubMed

    Moran, Corey S; Seto, Sai-Wang; Krishna, Smriti M; Sharma, Surabhi; Jose, Roby J; Biros, Erik; Wang, Yutang; Morton, Susan K; Golledge, Jonathan

    2017-02-21

    Intraluminal thrombus is a consistent feature of human abdominal aortic aneurysm (AAA). Coagulation factor Xa (FXa) catalyses FII to thrombin (FIIa). We examined the effect of FXa/FIIa inhibition on experimental aortic aneurysm in apolipoprotein E-deficient (ApoE(-/-)) mice infused with angiotensin II (AngII). The concentration of FXa within the supra-renal aorta (SRA) correlated positively with SRA diameter. Parenteral administration of enoxaparin (FXa/IIa inhibitor) and fondaparinux (FXa inhibitor) over 14 days reduced to severity of aortic aneurysm and atherosclerosis in AngII-infused ApoE(-/-) mice. Enteral administration of the FIIa inhibitor dabigatran had no significant effect. Aortic protease-activated receptor (PAR)-2 expression increased in response to AngII infusion. Fondaparinux reduced SRA levels of FXa, FIIa, PAR-2, matrix metalloproteinase (MMP)2, Smad2/3 phosphorylation, and MOMA-2 positive cells in the mouse model. FXa stimulated Smad2/3 phosphorylation and MMP2 expression in aortic vascular smooth muscle cells (VSMC) in vitro. Expression of MMP2 in FXa-stimulated VSMC was downregulated in the presence of a PAR-2 but not a PAR-1 inhibitor. These findings suggest that FXa/FIIa inhibition limits aortic aneurysm and atherosclerosis severity due to down-regulation of vascular PAR-2-mediated Smad2/3 signalling and MMP2 expression. Inhibition of FXa/FIIa may be a potential therapy for limiting aortic aneurysm.

  15. Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa.

    PubMed

    Huang, Xin; Dementiev, Alexey; Olson, Steven T; Gettins, Peter G W

    2010-06-25

    The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction approximately 2000-fold in the presence of phospholipid and Ca(2+). To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ(DeltaGD)) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing approximately 5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only approximately 2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional approximately 1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.

  16. Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

    SciTech Connect

    Huang, Xin; Dementiev, Alexey; Olson, Steven T.; Gettins, Peter G.W.

    2012-12-13

    The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction {approx}2000-fold in the presence of phospholipid and Ca{sup 2+}. To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ{sub {Delta}GD}) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing {approx}5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only {approx}2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional {approx}1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.

  17. Contemporary developments in the discovery of selective factor Xa inhibitors: A review.

    PubMed

    Patel, Nirav R; Patel, Dushyant V; Murumkar, Prashant R; Yadav, Mange Ram

    2016-10-04

    Thrombosis is a leading cause of death in cardiovascular diseases such as myocardial infarction (MI), unstable angina and acute coronary syndrome (ACS) in the industrialized world. Venous thromboembolism is observed in about 1 million people every year in United States causing significant morbidity and mortality. Conventional antithrombotic therapy has been reported to have several disadvantages and limitations like inconvenience in oral administration, bleeding risks (heparin analogs), narrow therapeutic window and undesirable interactions with food and drugs (vitamin K antagonist-warfarin). The unmet medical demand for orally active safe anticoagulants has generated widespread interest among the medicinal chemists engaged in this field. To modulate blood coagulation, various enzymes involved in the coagulation process have received great attention as potential targets by various research groups for the development of oral anticoagulants. Among these enzymes, factor Xa (FXa) has remained the centre of attention in the last decade. Intensive research efforts have been made by various research groups for the development of small, safe and orally bioavailable FXa inhibitors. This review is an attempt to compile the research work of various researchers in the direction of development of FXa inhibitors reported since 2010 onward.

  18. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    NASA Astrophysics Data System (ADS)

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-07-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

  19. Effect of famotidine on the pharmacokinetics of apixaban, an oral direct factor Xa inhibitor

    PubMed Central

    Upreti, Vijay V; Song, Yan; Wang, Jessie; Byon, Wonkyung; Boyd, Rebecca A; Pursley, Janice M; LaCreta, Frank; Frost, Charles E

    2013-01-01

    Background Apixaban is an oral, selective, direct factor Xa inhibitor approved for thromboprophylaxis after orthopedic surgery and stroke prevention in patients with atrial fibrillation, and under development for treatment of venous thromboembolism. This study investigated the effect of a gastric acid suppressant, famotidine (a histamine H2-receptor antagonist), on the pharmacokinetics of apixaban in healthy subjects. Methods This two-period, two-treatment crossover study randomized 18 healthy subjects to receive a single oral dose of apixaban 10 mg with and without a single oral dose of famotidine 40 mg administered 3 hours before dosing with apixaban. Plasma apixaban concentrations were measured up to 60 hours post-dose and pharmacokinetic parameters were calculated. Results Famotidine did not affect maximum apixaban plasma concentration (Cmax) or area under the plasma concentration-time curve from zero to infinite time (AUC∞). Point estimates for ratios of geometric means with and without famotidine were close to unity for Cmax (0.978) and AUC∞ (1.007), and 90% confidence intervals were entirely contained within the 80%–125% no-effect interval. Administration of apixaban alone and with famotidine was well tolerated. Conclusion Famotidine does not affect the pharmacokinetics of apixaban, consistent with the physicochemical properties of apixaban (lack of an ionizable group and pH-independent solubility). Apixaban pharmacokinetics would not be affected by an increase in gastrointestinal pH due to underlying conditions (eg, achlorhydria), or by gastrointestinal pH-mediated effects of other histamine H2-receptor antagonists, antacids, or proton pump inhibitors. Given that famotidine is also an inhibitor of the human organic cation transporter (hOCT), these results indicate that apixaban pharmacokinetics are not influenced by hOCT uptake transporter inhibitors. Overall, these results support that apixaban can be administered without regard to coadministration

  20. Potent Direct Inhibitors of Factor Xa Based on the Tetrahydroisoquinoline Scaffold

    PubMed Central

    Al-Horani, Rami A.; Mehta, Akul Y.; Desai, Umesh R.

    2012-01-01

    Direct inhibition of coagulation factor Xa (FXa) carries significant promise for developing effective and safe anticoagulants. Although a large number of FXa inhibitors have been studied, each can be classified as either possessing a highly flexible or a rigid core scaffold. We reasoned that an intermediate level of flexibility will provide high selectivity for FXa considering that its active site is less constrained in comparison to thrombin and more constrained as compared to trypsin. We studied several core scaffolds including 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid for direct FXa inhibition. Using a genetic algorithm-based docking and scoring approach, a promising candidate 23 was identified, synthesized, and found to inhibit FXa with a Ki of 28 μM. Optimization of derivative 23 resulted in the design of a potent dicarboxamide 47, which displayed a Ki of 135 nM. Dicarboxamide 47 displayed at least 1852-fold selectivity for FXa inhibition over other coagulation enzymes and doubled PT and aPTT of human plasma at 17.1 μM and 20.2 μM, respectively, which are comparable to those of clinically relevant agents. Dicarboxamide 47 is expected to serve as an excellent lead for further anticoagulant discovery. PMID:22770607

  1. Structure-based design of novel guanidine/benzamidine mimics: potent and orally bioavailable factor Xa inhibitors as novel anticoagulants.

    PubMed

    Lam, Patrick Y S; Clark, Charles G; Li, Renhua; Pinto, Donald J P; Orwat, Michael J; Galemmo, Robert A; Fevig, John M; Teleha, Christopher A; Alexander, Richard S; Smallwood, Angela M; Rossi, Karen A; Wright, Matthew R; Bai, Stephen A; He, Kan; Luettgen, Joseph M; Wong, Pancras C; Knabb, Robert M; Wexler, Ruth R

    2003-10-09

    As part of an ongoing effort to prepare orally active factor Xa inhibitors using structure-based drug design techniques and molecular recognition principles, a systematic study has been performed on the pharmacokinetic profile resulting from replacing the benzamidine in the P1 position with less basic benzamidine mimics or neutral residues. It is demonstrated that lowering the pK(a) of the P1 ligand resulted in compounds (3-benzylamine, 15a; 1-aminoisoquinoline, 24a; 3-aminobenzisoxazole, 23a; 3-phenylcarboxamide, 22b; and 4-methoxyphenyl, 22a) with improved pharmacokinetic features mainly as a result of decreased clearance, increased volume of distribution, and enhanced oral absorption. This work resulted in a series of potent and orally bioavailable factor Xa inhibitors that ultimately led to the discovery of SQ311, 24a. SQ311, which utilizes a 1-aminoisoquinoline as the P1 ligand, inhibits factor Xa with a K(i) of 0.33 nM and demonstrates both good in vivo antithrombotic efficacy and oral bioavailability.

  2. Population Pharmacokinetic and Pharmacodynamic Modeling Analysis of GCC‐4401C, a Novel Direct Factor Xa Inhibitor, in Healthy Volunteers

    PubMed Central

    Choi, HY; Choi, S; Kim, YH

    2016-01-01

    GCC‐4401C, an orally active direct factor Xa inhibitor that is similar to rivaroxaban, is currently under development for venous thromboembolic disease (VTE). The purpose of this study was to characterize the pharmacokinetics (PKs) and pharmacodynamics (PDs) of GCC‐4401C by population modeling analysis and to predict proper dosage regimens compared to rivaroxaban using data from two phase I clinical studies. Plasma GCC‐4401C concentrations over time were best described by a two‐compartment linear model and body weight was associated with central volume of distribution. Relevant PD markers generally changed in a dose‐dependent manner and were described well with sigmoid, simple maximum effect, or linear models. GCC‐4401C was absorbed more rapidly than rivaroxaban. Comparisons based on simulations of PD marker changes over time suggest that 20 mg and 40 mg of GCC‐4401C administered under fasted status are comparable to 10 mg and 20 mg of rivaroxaban under fed status. PMID:27511836

  3. Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa.

    PubMed

    Iyer, Janaki Krishnamoorthy; Otvos, Reka A; Kool, Jeroen; Kini, R Manjunatha

    2016-02-01

    Thrombin and factor Xa (FXa) are critical enzymes of the blood coagulation cascade and are excellent targets of anticoagulant agents. Natural sources present an array of anticoagulants that can be developed as antithrombotic drugs. High-resolution, online screening techniques have been developed for the identification of drug leads from complex mixtures. In this study, we have developed and optimized a microfluidic online screening technique coupled to nano-liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are suitable for the identification of inhibitors from sample amounts as low as sub-microliter volumes.

  4. Discovery of imidazo[1,5-c]imidazol-3-ones: weakly basic, orally active factor Xa inhibitors.

    PubMed

    Imaeda, Yasuhiro; Kuroita, Takanobu; Sakamoto, Hiroki; Kawamoto, Tetsuji; Tobisu, Mamoru; Konishi, Noriko; Hiroe, Katsuhiko; Kawamura, Masaki; Tanaka, Toshimasa; Kubo, Keiji

    2008-06-26

    The coagulation enzyme factor Xa (FXa) has been recognized as a promising target for the development of new antithrombotic agents. We previously found compound 1 to be an orally bioavailable FXa inhibitor in fasted monkeys; however, 1 showed poor bioavailability in rats and fed monkeys. To work out the pharmacokinetic problems, we focused our synthetic efforts on the chemical conversion of the 4-(imidazo[1,2- a]pyridin-5-yl)piperazine moiety of 1 to imidazolylpiperidine derivatives (fused and nonfused), which resulted in the discovery of the weakly basic imidazo[1,5- c]imidazol-3-one 3q as a potent and selective FXa inhibitor. Compound 3q showed favorable oral bioavailability in rats and monkeys under both fasted and fed conditions and antithrombotic efficacy in a rat model of venous thrombosis after oral administration, without a significant increase in bleeding time (unlike warfarin). On the basis of these promising properties, compound 3q was selected for further evaluation.

  5. Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds.

    PubMed

    Oliva, M L; Andrade, S A; Batista, I F; Sampaio, M U; Juliano, M; Fritz, H; Auerswald, E A; Sampaio, C A

    1999-12-01

    Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with Ki values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (Ki = 80 nM). The comparison between BuXI and BvTI reactive site structure indicates differences at Met59, Thr66 and Met67 residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (Km = 0.68 microM, k(cat)/Km = 1.3 x 10(6) M(-1) s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (Km = 0.66 microM, k(cat)/Km = 2.2 x 10(3) M(-1) s(-1)). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. The determined Km and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes.

  6. The effects of direct factor Xa inhibitor (Rivaroxaban) on the human osteoblastic cell line SaOS2.

    PubMed

    Gigi, Roy; Salai, Moshe; Dolkart, Oleg; Chechik, Ofir; Katzburg, Sarah; Stern, Naftali; Somjen, Dalia

    2012-01-01

    Thromboprophylaxis reduces the risk of surgery-related deep vein thrombosis, but anticoagulants were associated with systemic osteoporosis, a known risk factor for poor fracture healing. Rivaroxaban (XARELTO(®)) is a novel anticoagulant with specific ability to inhibit factor Xa, a serine endopeptidase, which plays a key role in coagulation. This study investigated the direct effects of rivaroxaban on bone biology using an in vitro cell culture model from the human female osteoblastic cell line SaOS2. Cells at subconfluence were treated for 24 hr with different concentrations of rivaroxaban and analyzed for DNA synthesis and creatine kinase- and alkaline phosphatase-specific activities, and were treated 21 days for analyzing mineralization. Rivaroxaban (0.01-50 μg/ml) dose-dependently inhibited up to 60% DNA synthesis of the cells. Creatine kinase-specific activity was also inhibited dose-dependently to a similar extent by the same concentrations. Alkaline phosphatase-specific activity was dose-dependently inhibited but only up to 30%. Cell mineralization was unaffected by 10 μg/ml rivaroxaban. This model demonstrated a significant rivaroxaban-induced reduction in osteoblastic cell growth and energy metabolism, and slight inhibition of the osteoblastic marker, alkaline phosphatase, while osteoblastic mineralization was unaffected. These findings might indicate that rivaroxaban inhibits the first stage of bone formation but does not affect later stages (i.e., bone mineralization).

  7. CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation.

    PubMed

    Salu, Bruno R; Ferreira, Rodrigo S; Brito, Marlon V; Ottaiano, Tatiana F; Cruz, José Walber M C; Silva, Mariana Cristina C; Correia, Maria Tereza S; Paiva, Patrícia M G; Maffei, Francisco Humberto A; Oliva, Maria Luiza Vilela

    2014-09-01

    Arterial thrombosis is an important complication of diabetes and cancer, being an important target for therapeutic intervention. Crataeva tapia bark lectin (CrataBL) has been previously shown to have hypoglycemiant effect and also to induce cancer cell apoptosis. It also showed inhibitory activity against Factor Xa (Kiapp=8.6 μm). In the present study, we evaluated the anti-thrombotic properties of CrataBL in arterial thrombosis model. CrataBL prolongs the activated partial thromboplastin time on human and mouse plasma, and it impairs the heparin-induced potentiation of antithrombin III and heparin-induced platelet activation in the presence of low-dose ADP. It is likely that the dense track of positive charge on CrataBL surface competes with the heparin ability to bind to antithrombin III and to stimulate platelets. In the photochemically induced thrombosis model in mice, in the groups treated with 1.25, 5.0, or 10 mg/kg CrataBL, prior to the thrombus induction, the time of total artery occlusion was prolonged by 33.38%, 65%, and 66.11%, respectively, relative to the time of the control group. In contrast to heparin, the bleeding time in CrataBL-treated mice was no longer than in the control. In conclusion, CrataBL was effective in blocking coagulation and arterial thrombus formation, without increasing bleeding time.

  8. Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions.

    PubMed

    Kim, Tae Kwon; Tirloni, Lucas; Radulovic, Zeljko; Lewis, Lauren; Bakshi, Mariam; Hill, Creston; da Silva Vaz, Itabajara; Logullo, Carlos; Termignoni, Carlos; Mulenga, Albert

    2015-08-01

    Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin 19 that is characterised by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterisation reveals that the ubiquitously transcribed A. americanum serpin 19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) A. americanum serpin 19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, recombinant A. americanum serpin 19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of recombinant A. americanum serpin 19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14-29%. In anti-hemostatic functional assays, recombinant A. americanum serpin 19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250s, and up to 40s in the activated partial thromboplastin time assay. Given A. americanum serpin 19 high cross-tick species conservation, and specific reactivity of recombinant A. americanum serpin 19 with antibodies to A. americanum tick saliva proteins, we conclude that recombinant A. americanum serpin 19 is a potential candidate for development of a universal tick vaccine.

  9. The discovery of (2R,4R)-N-(4-chlorophenyl)-N- (2-fluoro-4-(2-oxopyridin-1(2H)-yl)phenyl)-4-methoxypyrrolidine-1,2-dicarboxamide (PD 0348292), an orally efficacious factor Xa inhibitor.

    PubMed

    Kohrt, Jeffrey T; Bigge, Christopher F; Bryant, John W; Casimiro-Garcia, Agustin; Chi, Liguo; Cody, Wayne L; Dahring, Tawny; Dudley, Danette A; Filipski, Kevin J; Haarer, Staci; Heemstra, Ron; Janiczek, Nancy; Narasimhan, Lakshmi; McClanahan, Thomas; Peterson, J Thomas; Sahasrabudhe, Vaisheli; Schaum, Robert; Van Huis, Chad A; Welch, Kathleen M; Zhang, Erli; Leadley, Robert J; Edmunds, Jeremy J

    2007-08-01

    Herein, we report the discovery of novel, proline-based factor Xa inhibitors containing a neutral P1 chlorophenyl pharmacophore. Through the additional incorporation of 1-(4-amino-3-fluoro-phenyl)-1H-pyridin-2-one 22, as a P4 pharmacophore, we discovered compound 7 (PD 0348292). This compound is a selective, orally bioavailable, efficacious FXa inhibitor that is currently in phase II clinical trials for the treatment and prevention of thrombotic disorders.

  10. Lufaxin, a Novel Factor Xa Inhibitor from the Salivary Gland of the sand fly Lutzomyia longipalpis, Blocks PAR2 Activation and Inhibits Inflammation and Thrombosis in Vivo

    PubMed Central

    Collin, Nicolas; Assumpção, Teresa C. F.; Mizurini, Daniella M.; Gilmore, Dana; Dutra-Oliveira, Angélica; Kotsyfakis, Michalis; Sá-Nunes, Anderson; Teixeira, Clarissa; Ribeiro, José M. C.; Monteiro, Robson Q.; Valenzuela, Jesus G.; Francischetti, Ivo M. B.

    2012-01-01

    Objective Blood-sucking arthropods salivary glands (SGs) contain a remarkable diversity of antihemostatics. The aim of this study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results Several L. longipalpis salivary proteins were expressed in HEK293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, non-competitive, and reversible inhibitor of Factor Xa (FXa). Notably, Lufaxin primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, DEGR- FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both PT and aPTT. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with a KD ~3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents PAR2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs aPTT ex vivo, implying that it works as an anticoagulant in vivo. Finally, SG of sandflies was found to inhibit FXa and to interact with the enzyme. Conclusion Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and antiinflamatory activities. It is a useful tool to understand FXa structural features and its role in pro-hemostatic and pro-inflammatory events. PMID:22796577

  11. A computational modeling and molecular dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)

    PubMed Central

    Chandrasekaran, Vasudevan; Lee, Chang Jun; Lin, Ping; Duke, Robert E.

    2009-01-01

    Protein Z-dependent protease inhibitor (ZPI) and antithrombin III (AT3) are members of the serpin superfamily of protease inhibitors that inhibit factor Xa (FXa) and other proteases in the coagulation pathway. While experimental structural information is available for the interaction of AT3 with FXa, at present there is no structural data regarding the interaction of ZPI with FXa, and the precise role of this interaction in the blood coagulation pathway is poorly understood. In an effort to gain a structural understanding of this system, we have built a solvent equilibrated three-dimensional structural model of the Michaelis complex of human ZPI/FXa using homology modeling, protein–protein docking and molecular dynamics simulation methods. Preliminary analysis of interactions at the complex interface from our simulations suggests that the interactions of the reactive center loop (RCL) and the exosite surface of ZPI with FXa are similar to those observed from X-ray crystal structure-based simulations of AT3/FXa. However, detailed comparison of our modeled structure of ZPI/FXa with that of AT3/FXa points to differences in interaction specificity at the reactive center and in the stability of the inhibitory complex, due to the presence of a tyrosine residue at the P1 position in ZPI, instead of the P1 arginine residue in AT3. The modeled structure also shows specific structural differences between AT3 and ZPI in the heparin-binding and flexible N-terminal tail regions. Our structural model of ZPI/FXa is also compatible with available experimental information regarding the importance for the inhibitory action of certain basic residues in FXa. PMID:19172319

  12. The effect of the direct factor Xa inhibitors apixaban and rivaroxaban on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples.

    PubMed

    Bonar, Roslyn; Favaloro, Emmanuel J; Mohammed, Soma; Ahuja, Monica; Pasalic, Leonardo; Sioufi, John; Marsden, Katherine

    2016-01-01

    The direct oral anticoagulants (DOACs), now including dabigatran, apixaban and rivaroxaban, have given clinicians alternative options to low molecular weight heparins (LMWHs) and vitamin K antagonist therapy, including warfarin, for the treatment of atrial fibrillation and treatment and prevention of venous thromboembolic (VTE) disease. DOACs have been successfully marketed as not requiring monitoring; however, there will be situations where clinicians will request laboratory testing, including emergency department admissions for haemorrhage or thrombosis, or emergency surgical interventions. We report the results of several Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) surveys using apixaban and rivaroxaban spiked samples to either assess the suitability of certain potential screening or drug-quantifying assays, for assessment of drug presence or absence or measurement of levels, as well as assessing potential interference in a wide variety of haemostasis assays. We also include additional evaluations using ex vivo samples from patients given apixaban and rivaroxaban for various therapeutic reasons. The prothrombin time (PT) and activated partial thromboplastin time (APTT) show better sensitivity with rivaroxaban than apixaban. Anti-Xa assays show good concordance and reproducibility with expected drug levels; however, availability of these assays may be limited to larger institutions. Interference of apixaban and rivaroxaban on haemostasis testing extends beyond routine coagulation assays to encompass a plethora of specialised assays, including factor assays, lupus inhibitor, and FVIII inhibitor estimation. In conclusion, this report highlights the influence of these drugs on most tests performed in haemostasis laboratories, and the potential for some tests to predict the presence, absence or level of these drugs in plasma.

  13. Rivaroxaban, a direct inhibitor of the coagulation factor Xa interferes with hormonal-induced physiological modulations in human female osteoblastic cell line SaSO2.

    PubMed

    Somjen, Dalia; Katzburg, Sara; Gigi, Roi; Dolkart, Oleg; Sharon, Orli; Salai, Moshe; Stern, Naftali

    2013-05-01

    The use of anticoagulants has been associated with systemic osteoporosis and increased risk for poor fracture healing but is inevitable following major orthopedic surgery of lower limbs. Rivaroxaban A (R) is an anticoagulant recently introduced in the clinical setting, which is a specific factor Xa inhibitor. We reported previously that R significantly inhibited cell growth, energy metabolism and alkaline phosphatase activity in human osteoblastic cell line SaOS2, with no effect on mineralization, indicating transient inhibition of bone formation. We now investigated the effects of R on SaOS2 response to osteoblast-modulating hormones. At sub-confluence cells were treated with: estradiol-17β (E2), the phytoestrogens daidzein (D) and biochainin A (BA), the carboxy-pytoestrogenic derivative carboxy-D (cD), the estrogen receptor α (ERα) agonist PPT, the estrogen receptor β (ERβ) agonist DPN, parathyroid hormone (PTH) and several vitamin D metabolites and analogs with/without R for 24h. All hormones tested stimulated significantly DNA synthesis (DNA), creatine kinase (CK) and alkaline phosphatase (ALP) specific activities, but all these stimulations were totally inhibited when given together with R. R had no effect on mRNA expression of ERα, ERβ and 25 Hydroxy-vitamin D3-1α hydroxylase (1OHase), but inhibited hormonal modulations of mRNA expressions. In conclusion R inhibited significantly hormonal stimulation of different parameters indicating inhibition of not only the early stages of bone formation, but also the stimulatory effects of bone modulating hormones with a yet unclear mechanism. The relevance of these findings to human bone physiology is yet to be investigated.

  14. Home treatment of patients with low-risk pulmonary embolism with the oral factor Xa inhibitor rivaroxaban. Rationale and design of the HoT-PE Trial.

    PubMed

    Barco, Stefano; Lankeit, Mareike; Binder, Harald; Schellong, Sebastian; Christ, Michael; Beyer-Westendorf, Jan; Duerschmied, Daniel; Bauersachs, Rupert; Empen, Klaus; Held, Matthias; Schwaiblmair, Martin; Fonseca, Cândida; Jiménez, David; Becattini, Cecilia; Quitzau, Kurt; Konstantinides, Stavros

    2016-07-04

    Pulmonary embolism (PE) is a potentially life-threatening acute cardiovascular syndrome. However, more than 95 % of patients are haemodynamically stable at presentation, and among them are patients at truly low risk who may qualify for immediate or early discharge. The Home Treatment of Pulmonary Embolism (HoT-PE) study is a prospective international multicentre single-arm phase 4 management (cohort) trial aiming to determine whether home treatment of acute low-risk PE with the oral factor Xa inhibitor rivaroxaban is feasible, effective, and safe. Patients with confirmed PE, who have no right ventricular dysfunction or free floating thrombi in the right atrium or ventricle, are eligible if they meet none of the exclusion criteria indicating haemodynamic instability, serious comorbidity or any condition mandating hospitalisation, or a familial/social environment unable to support home treatment. The first dose of rivaroxaban is given in hospital, and patients are discharged within 48 hours of presentation. Rivaroxaban is taken for at least three months. The primary outcome is symptomatic recurrent venous thromboembolism or PE-related death within three months of enrolment. Secondary outcomes include quality of life and patient satisfaction, and health care resource utilisation compared to existing data on standard-duration hospital treatment. HoT-PE is planned to analyse 1,050 enrolled patients, providing 80 % power to reject the null hypothesis that the recurrence rate of venous thromboembolism is >3 % with α≤0.05. If the hypothesis of HoT-PE is confirmed, early discharge and out-of-hospital treatment may become an attractive, potentially cost-saving option for a significant proportion of patients with acute PE.

  15. At-line nanofractionation with parallel mass spectrometry and bioactivity assessment for the rapid screening of thrombin and factor Xa inhibitors in snake venoms.

    PubMed

    Mladic, Marija; Zietek, Barbara M; Iyer, Janaki Krishnamoorthy; Hermarij, Philip; Niessen, Wilfried M A; Somsen, Govert W; Kini, R Manjunatha; Kool, Jeroen

    2016-02-01

    Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease

  16. Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR{sub 1} activation

    SciTech Connect

    Blanc-Brude, Olivier P. . E-mail: olivier.blanc-brude@larib.inserm.fr; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

    2005-03-10

    Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR{sub 1}). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR{sub 1}-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR{sub 1}-specific agonists and inhibitors were used to demonstrate that PAR{sub 1} mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR{sub 1} and not PAR{sub 2}. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.

  17. Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

    PubMed

    Pinto, D J; Orwat, M J; Wang, S; Fevig, J M; Quan, M L; Amparo, E; Cacciola, J; Rossi, K A; Alexander, R S; Smallwood, A M; Luettgen, J M; Liang, L; Aungst, B J; Wright, M R; Knabb, R M; Wong, P C; Wexler, R R; Lam, P Y

    2001-02-15

    Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.

  18. Comparative evaluation of direct thrombin and factor Xa inhibitors with antiplatelet agents under flow and static conditions: an in vitro flow chamber model.

    PubMed

    Hosokawa, Kazuya; Ohnishi, Tomoko; Sameshima, Hisayo; Miura, Naoki; Koide, Takehiko; Maruyama, Ikuro; Tanaka, Kenichi A

    2014-01-01

    Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s(-1). Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.

  19. 1-[3-Aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa.

    PubMed

    Pinto, Donald J P; Orwat, Michael J; Quan, Mimi L; Han, Qi; Galemmo, Robert A; Amparo, Eugene; Wells, Brian; Ellis, Christopher; He, Ming Y; Alexander, Richard S; Rossi, Karen A; Smallwood, Angela; Wong, Pancras C; Luettgen, Joseph M; Rendina, Alan R; Knabb, Robert M; Mersinger, Lawrence; Kettner, Charles; Bai, Steven; He, Kan; Wexler, Ruth R; Lam, Patrick Y S

    2006-08-01

    Attempts to further optimize the pyrazole factor Xa inhibitors centered on masking the aryl aniline P4 moiety. Scaffold optimization resulted in the identification of a novel bicyclic pyrazolo-pyridinone scaffold which retained fXa potency. The novel bicyclic scaffold preserved all binding interactions observed with the monocyclic counterpart and importantly the carboxamido moiety was integrated within the scaffold making it less susceptible to hydrolysis. These efforts led to the identification of 1-[3-aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one 6f (BMS-740808), a highly potent (fXa Ki=30 pM) with a rapid onset of inhibition (2.7x10(7) M-1 s-1) in vitro, selective (>1000-fold over other proteases), efficacious in the AVShunt thrombosis model, and orally bioavailable inhibitor of blood coagulation factor Xa.

  20. [Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors. Proposals of the Working Group on Perioperative Haemostasis (GIHP) - March 2013].

    PubMed

    Pernod, G; Albaladejo, P; Godier, A; Samama, C M; Susen, S; Gruel, Y; Blais, N; Fontana, P; Cohen, A; Llau, J V; Rosencher, N; Schved, J F; de Maistre, E; Samama, M M; Mismetti, P; Sié, P

    2013-10-01

    New direct oral anticoagulants (NOAC), inhibitors of factor IIa or Xa, are expected to be widely used for the treatment of venous thromboembolic disease, or in case of atrial fibrillation. Such anticoagulant treatments are known to be associated with haemorrhagic complications. Moreover, it is likely that such patients on long-term treatment with NOAC will be exposed to emergency surgery or invasive procedures. Due to the present lack of experience in such conditions, we cannot make recommendations, but only propose management for optimal safety as regards the risk of bleeding in such emergency conditions. In this article, only dabigatran and rivaroxaban were discussed. For emergency surgery at risk of bleeding, we propose to dose the plasmatic concentration of drug. Levels inferior or equal to 30ng/mL for both dabigatran and rivaroxaban, should enable the realization of a high bleeding risk surgery. For higher concentration, it was proposed to postpone surgery by monitoring the evolution of the drug concentration. Action is then defined by the kind of NOAC and its concentration. If the dosage of the drug is not immediately available, proposals only based on the usual tests, PT and aPTT, also are presented. However, these tests do not really assess drug concentration or bleeding risk. In case of severe haemorrhage in a critical organ, it is proposed to reduce the effect of anticoagulant therapy using a nonspecific procoagulant drug (activated prothrombin concentrate, FEIBA, 30-50U/kg, or non-activated 4-factors prothrombin concentrates 50U/kg). For any other type of severe haemorrhage, the administration of such a procoagulant drug, potentially thrombogenic in these patients, will be discussed regarding concentration of NACO and possibilities for mechanical haemostasis.

  1. Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors: proposals of the working group on perioperative haemostasis (GIHP) - March 2013.

    PubMed

    Pernod, Gilles; Albaladejo, Pierre; Godier, Anne; Samama, Charles M; Susen, Sophie; Gruel, Yves; Blais, Normand; Fontana, Pierre; Cohen, Ariel; Llau, Juan V; Rosencher, Nadia; Schved, Jean-François; de Maistre, Emmanuel; Samama, Meyer M; Mismetti, Patrick; Sié, Pierre

    2013-01-01

    Direct new oral anticoagulants (NOACs) - inhibitors of thrombin or factor Xa - are intended to be used largely in the treatment of venous thromboembolic disease or the prevention of systematic embolism in atrial fibrillation, instead of vitamin K antagonists. Like any anticoagulant treatment, they are associated with spontaneous or provoked haemorrhagic risk. Furthermore, a significant proportion of treated patients are likely to be exposed to emergency surgery or invasive procedures. Given the absence of a specific antidote, the action to be taken in these situations must be defined. The lack of data means that it is only possible to issue proposals rather than recommendations, which will evolve according to accumulated experience. The proposals presented here apply to dabigatran (Pradaxa(®)) and rivaroxaban (Xarelto(®)); data for apixaban and edoxaban are still scarce. For urgent surgery with haemorrhagic risk, the drug plasma concentration should be less or equal to 30ng/mL for dabigatran and rivaroxaban should enable surgery associated with a high bleeding risk. Beyond that, if possible, the intervention should be postponed by monitoring the drug concentration. The course to follow is then defined according to the NOAC and its concentration. If the anticoagulant dosage is not immediately available, worse propositions, based on the usual tests (prothrombin time and activated partial thromboplastin time), are presented. However, these tests do not really assess drug concentration or the risk of bleeding that depends on it. In case of serious bleeding in a critical organ, the effect of anticoagulant therapy should be reduced using a non-specific procoagulant drug as a first-line approach: activated prothrombin complex concentrate (aPCC) (FEIBA(®) 30-50U/kg) or non-activated PCC (50U/kg). In addition, for any other type of severe haemorrhage, the administration of a procoagulant drug, which is potentially thrombogenic in these patients, is discussed according

  2. Future therapeutic directions for factor Xa inhibition in the prophylaxis and treatment of thrombotic disorders.

    PubMed

    Turpie, Alexander G G

    2003-11-15

    The targeted mechanism of factor Xa inhibition has been studied extensively, initially as prophylaxis for venous thromboembolism (VTE) in the orthopedic surgical setting. Future therapeutic directions for selective factor Xa inhibition in the management of other thrombotic diseases are discussed. Thromboembolic diseases can occur in the venous or arterial sides of the circulatory system. Factor Xa inhibition is a targeted approach to anticoagulation that resulted from significant advances in our understanding of the coagulation cascade. The factor Xa inhibitor fondaparinux has been studied extensively in the orthopedic surgical setting for the prophylaxis of VTE. Current investigations that are under way or completed evaluate the efficacy and safety of fondaparinux for the management of various thrombotic diseases. The future development of fondaparinux resides primarily in three therapeutic areas: prevention of VTE, treatment of VTE, and treatment of acute coronary syndromes. For the prevention of VTE, fondaparinux has been studied as extended prophylaxis following hip fracture surgery (PENTHIFRA Plus), for use in high-risk abdominal surgical patients (PEGASUS and APOLLO), and for use in medical patients (ARTEMIS). Studies evaluating fondaparinux for the treatment of VTE are part of the large MATISSE clinical program (MATISSE DVT and MATISSE PE). Fondaparinux was investigated in phase 2 studies for the treatment of acute coronary syndromes, including acute ST-segment myocardial infarction (PENTALYSE) and unstable angina (PENTUA). Encouraging data from these trials are the basis for phase 3 programs in this area (MICHELANGELO). The orthopedic prophylactic and nonorthopedic clinical programs for fondaparinux in the management of thrombosis support the concept that targeted inhibition of coagulation is an effective advance in antithrombotic therapy.

  3. Clinical decision-making for vitamin K-1 and K-2 deficiency and coronary artery calcification with warfarin therapy: are diet, factor Xa inhibitors or both the answer?

    PubMed

    Wahlqvist, Mark L; Tanaka, Kiyoshi; Tzeng, Bing-Hsiean

    2013-01-01

    Coronary artery calcification is a recognised risk factor for ischaemic heart disease and mortality. Evidence is now strong that Mönckeberg's arteriosclerosis, a form of vascular calcification, can be attributable to vitamin K deficiency, but that vitamin K-2, especially the MK-4 form from foods like cheese can be protective. Warfarin blocks the recycling of hepatic and peripheral vitamin K leading to secondary vitamin K deficiency with adverse effects on vasculature, bone, kidneys, brain and other tissues and systems (inflammatory, immune function and neoplasia at least). There is individual susceptibility to vitamin K deficiency and warfarin sensitivity, partly explicable in terms of genetic polymorphisms, epigenetics, diet and pharmacotherapy. The emergence of extensive coronary calcification in a man with atrial fibrillation treated for a decade with warfarin is described by way of illustration and to raise the present clinical management conundrums. Finally, a putative set of recommendations is provided.

  4. The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

    PubMed Central

    Figueiredo, Ana C.; de Sanctis, Daniele; Pereira, Pedro José Barbosa

    2013-01-01

    The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin. PMID:23951260

  5. The relative molecular mass dependence of the anti-factor Xa properties of heparin.

    PubMed Central

    Ellis, V; Scully, M F; Kakkar, V V

    1986-01-01

    The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 X 10(7) M-1 X min-1, compared with 6.3 X 10(4) M-1 X min-1 in the absence of heparin, and this rose progressively to 4.2 X 10(8) M-1 X min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III. PMID:3800942

  6. Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

    SciTech Connect

    Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian . E-mail: bzheng@hkucc.hku.hk

    2007-07-20

    The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.

  7. Stabilizing of a globular protein by a highly complex water network: a molecular dynamics simulation study on factor Xa.

    PubMed

    Wallnoefer, Hannes G; Handschuh, Sandra; Liedl, Klaus R; Fox, Thomas

    2010-06-03

    The role of water molecules is increasingly attracting attention in structural biology, and many studies have demonstrated their crucial contribution to the stability and function of proteins. Here, we present molecular dynamics studies on factor Xa (fXa) to investigate the effect of water molecules in this serine protease. fXa is a key enzyme in the blood coagulation cascade, and thus, an important target for antithrombotic drugs. A reasonable representation of the structure is crucial for an investigation at the molecular level and, thus, a prerequisite for structure-based drug design. Simulations of well-resolved fXa X-ray structures with different sets of water molecules show the importance of a well-determined water set for the simulation. We discuss implications of different water sets on the structure and dynamics of fXa.

  8. Cerebral Venous Thromboembolism in Antiphospholipid Syndrome Successfully Treated with the Combined Use of an Anti-Xa Inhibitor and Corticosteroid.

    PubMed

    Sugie, Masayuki; Iizuka, Natsuko; Shimizu, Yuki; Ichikawa, Hiroo

    2015-01-01

    We herein report a case presenting with cerebral venous sinus thrombosis (CVST) associated with primary antiphospholipid syndrome (APS). The patient developed recurrent CVST followed by a hemorrhagic ischemic stroke despite the use of warfarin during the appropriate therapeutic window. Thus, we substituted warfarin to rivaroxaban with prednisolone and obtained a good clinical course. In addition to the effect of prednisolone of inhibiting elevated lupus anticoagulants and the recurrence of arterial thrombosis, rivaroxaban may prevent CVST and inhibit hypercoagulability induced by corticosteroids. The combination of an anti-Xa inhibitor and corticosteroid may be an alternative treatment for CVST and arterial thrombus with warfarin-resistant APS.

  9. Phosphatidylserine-induced factor Xa dimerization and binding to factor Va are competing processes in solution.

    PubMed

    Majumder, Rinku; Koklic, Tilen; Rezaie, Alireza R; Lentz, Barry R

    2013-01-08

    A soluble, short chain phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to promote FXa dimerization (K(d) ~ 14 nM), and to enhance both the catalytic activity of FXa and the cofactor activity of FVa. In the presence of calcium, C6PS binds to two sites on FXa, one in the epidermal growth factor-like (EGF) domain and one in the catalytic domain; the latter interaction is sensitive to Na(+) binding and probably represents a protein recognition site. Here we ask whether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes. We monitored FXa activity at 5, 20, and 50 nM FXa while titrating with FVa in the presence of 400 μM C6PS and 3 or 5 mM Ca(2+) to show that the apparent K(d) of FVa-FXa interaction increased with an increase in FXa concentration at 5 mM Ca(2+), but the K(d) was only slightly affected at 3 mM Ca(2+). A mixture of 50 nM FXa and 50 nM FVa in the presence of 400 μM C6PS yielded both Xa homodimers and Xa·Va heterodimers, but no FXa dimers bound to FVa. A mutant FXa (R165A) that has reduced prothrombinase activity showed both weakened dimerization (K(d) ~ 147 nM) and weakened FVa binding (apparent K(d) values of 58, 92, and 128 nM for 5, 20, and 50 nM R165A FXa, respectively). Native gel electrophoresis showed that the GLA-EGF(NC) fragment of FXa (lacking the catalytic domain) neither dimerized nor formed a complex with FVa in the presence of 400 μM C6PS and 5 mM Ca(2+). Our results demonstrate that the dimerization site and FVa-binding site are both located in the catalytic domain of FXa and that these sites are linked thermodynamically.

  10. Discovery of 1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro- 1H-pyrazolo[3,4-c]pyridine-3-carboxamide (Apixaban, BMS-562247), a Highly Potent, Selective, Efficacious, and Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa

    SciTech Connect

    Pinto, Donald J.P.; Orwat, Michael J.; Koch, Stephanie; Rossi, Karen A.; Alexander, Richard S.; Smallwood, Angela; Wong, Pancras C.; Rendina, Alan R.; Luettgen, Joseph M.; Knabb, Robert M.; He, Kan; Xin, Baomin; Wexler, Ruth R.; Lam, Patrick Y.S.

    2010-03-08

    Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P{sub 1} moieties that resulted in the identification of the p-methoxyphenyl P{sub 1}, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P{sub 4} ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.

  11. Acylcarnitines are anticoagulants that inhibit factor Xa and are reduced in venous thrombosis, based on metabolomics data

    PubMed Central

    Deguchi, Hiroshi; Banerjee, Yajnavalka; Trauger, Sunia; Siuzdak, Gary; Kalisiak, Ewa; Fernández, José A.; Hoang, Linh; Tran, Minerva; Yegneswaran, Subramanian; Elias, Darlene J.

    2015-01-01

    In many patients with deep vein thrombosis and pulmonary embolism (venous thromboembolism, VTE), biomarkers or genetic risk factors have not been identified. To discover novel plasma metabolites associated with VTE risk, we employed liquid chromatography-mass spectrometry-based untargeted metabolomics, which do not target any specific metabolites. Using the Scripps Venous Thrombosis Registry population for a case-control study, we discovered that 10:1 and 16:1 acylcarnitines were low in plasmas of the VTE patient group compared with matched controls, respectively. Data from targeted metabolomics studies showed that several long-chain acylcarnitines (10:1, 12:0, 12:2, 18:1, and 18:2) were lower in the VTE group. Clotting assays were used to evaluate a causal relationship for low acylcarnitines in patients with VTE. Various acylcarnitines inhibited factor Xa-initiated clotting. Inhibition of factor Xa by acylcarnitines was greater for longer acyl chains. Mechanistic studies showed that 16:0 acylcarnitine had anticoagulant activity in the absence of factor Va or phospholipids. Surface plasmon resonance investigations revealed that 16:0 acylcarnitine was bound to factor Xa and that binding did not require the γ-carboxy glutamic acid domain. In summary, our study identified low plasma levels of acylcarnitines in patients with VTE and showed that acylcarnitines have anticoagulant activity related to an ability to bind and inhibit factor Xa. PMID:26175037

  12. Group D prothrombin activators from snake venom are structural homologues of mammalian blood coagulation factor Xa.

    PubMed Central

    Rao, Veena S; Joseph, Jeremiah S; Kini, R Manjunatha

    2003-01-01

    Procoagulant venoms of several Australian elapids contain proteinases that specifically activate prothrombin; among these, Group D activators are functionally similar to coagulation factor Xa (FXa). Structural information on this class of prothrombin activators will contribute significantly towards understanding the mechanism of FXa-mediated prothrombin activation. Here we present the purification of Group D prothrombin activators from three Australian snake venoms (Hoplocephalus stephensi, Notechis scutatus scutatus and Notechis ater niger) using a single-step method, and their N-terminal sequences. The N-terminal sequence of the heavy chain of hopsarin D (H. stephensi) revealed that a fully conserved Cys-7 was substituted with a Ser residue. We therefore determined the complete amino acid sequence of hopsarin D. Hopsarin D shows approximately 70% similarity with FXa and approximately 98% similarity with trocarin D, a Group D prothrombin activator from Tropidechis carinatus. It possesses the characteristic Gla domain, two epidermal growth factor-like domains and a serine proteinase domain. All residues important for catalysis are conserved, as are most regions involved in interactions with factor Va and prothrombin. However, there are some structural differences. Unlike FXa, hopsarin D is glycosylated in both its chains: in light-chain residue 52 and heavy-chain residue 45. The glycosylation on the heavy chain is a large carbohydrate moiety adjacent to the active-site pocket. Overall, hopsarin D is structurally and functionally similar to mammalian coagulation FXa. PMID:12403650

  13. A novel microfluidic anti-factor Xa assay device for monitoring anticoagulant therapy at the point-of-care

    NASA Astrophysics Data System (ADS)

    Harris, Leanne F.; Rainey, Paul; Castro-López, Vanessa; O'Donnell, James S.; Killard, Anthony J.

    2013-05-01

    Millions of patients worldwide are receiving anticoagulant therapy to treat hypercoagulable diseases. While standard testing is still performed in the central laboratory, point-of-care (POC) diagnostics are being developed due to the increasing number of patients requiring long-term anticoagulation and with a need for more personalized and targeted therapy. Many POC devices on the market focus on clot measurement, a technique which is limited in terms of variability, highlighting the need for more reliable assays of anticoagulant status. The anti-Xa assay, a factor specific optical assay, was developed to measure the extent to which exogenous factor Xa (FXa) is inhibited by heparinantithrombin complexes. We have developed a novel microfluidic device and assay for monitoring the effect of heparin anticoagulant therapy at the point-of-care. The assay which was also developed in our institute is based on the anti-Xa assay principle but uses fluorescence as the method of detection. Our device is a disposable laminate microfluidic strip, fabricated from the cyclic polyolefin (COP), Zeonor®, which is extremely suitable for application to fluorescent device platforms. We present data on the execution of the anti-Xa assay in this microfluidic format, demonstrating that the assay can be used to measure heparin in human plasma samples from 0 to 0.8 U/ml, with average assay reproducibility of 8% and a rapid result obtained within 60 seconds. Results indicate that with further development, the fluorogenic anti-Xa assay and device could become a successful method for monitoring anticoagulant therapy.

  14. Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

    PubMed

    Podoplelova, Nadezhda A; Sveshnikova, Anastasia N; Kurasawa, James H; Sarafanov, Andrey G; Chambost, Herve; Vasil'ev, Sergey A; Demina, Irina A; Ataullakhanov, Fazly I; Alessi, Marie-Christine; Panteleev, Mikhail A

    2016-06-01

    Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow.

  15. Protein-Templated Formation of an Inhibitor of the Blood Coagulation Factor Xa through a Background-Free Amidation Reaction.

    PubMed

    Jaegle, Mike; Steinmetzer, Torsten; Rademann, Jörg

    2017-03-20

    Protein-templated reactions enable the target-guided formation of protein ligands from reactive fragments, ideally with no background reaction. Herein, we investigate the templated formation of amides. A nucleophilic fragment that binds to the coagulation factor Xa was incubated with the protein and thirteen differentially activated dipeptides. The protein induced a non-catalytic templated reaction for the phenyl and trifluoroethyl esters; the latter was shown to be a completely background-free reaction. Starting from two fragments with millimolar affinity, a 29 nm superadditive inhibitor of factor Xa was obtained. The fragment ligation reaction was detected with high sensitivity by an enzyme activity assay and by mass spectrometry. The reaction progress and autoinhibition of the templated reaction by the formed ligation product were determined, and the structure of the protein-inhibitor complex was elucidated.

  16. Protein‐Templated Formation of an Inhibitor of the Blood Coagulation Factor Xa through a Background‐Free Amidation Reaction

    PubMed Central

    Jaegle, Mike; Steinmetzer, Torsten

    2017-01-01

    Abstract Protein‐templated reactions enable the target‐guided formation of protein ligands from reactive fragments, ideally with no background reaction. Herein, we investigate the templated formation of amides. A nucleophilic fragment that binds to the coagulation factor Xa was incubated with the protein and thirteen differentially activated dipeptides. The protein induced a non‐catalytic templated reaction for the phenyl and trifluoroethyl esters; the latter was shown to be a completely background‐free reaction. Starting from two fragments with millimolar affinity, a 29 nm superadditive inhibitor of factor Xa was obtained. The fragment ligation reaction was detected with high sensitivity by an enzyme activity assay and by mass spectrometry. The reaction progress and autoinhibition of the templated reaction by the formed ligation product were determined, and the structure of the protein–inhibitor complex was elucidated. PMID:28199769

  17. The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides.

    PubMed Central

    Barrowcliffe, T W; Havercroft, S J; Kemball-Cook, G; Lindahl, U

    1987-01-01

    The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results. PMID:3606581

  18. Effects of factor Xa on the expression of proteins in femoral arteries from type 2 diabetic patients

    PubMed Central

    López-Farré, Antonio J; Rodriguez-Sierra, Pablo; Modrego, Javier; Segura, Antonio; Martín-Palacios, Naiara; Saiz, Ana M; Zamorano-León, José J; Duarte, Juan; Serrano, Javier; Moñux, Guillermo

    2014-01-01

    Aim Further to its pivotal role in haemostasis, factor Xa (FXa) promotes effects on the vascular wall. The purpose of the study was to evaluate if FXa modifies the expression level of energy metabolism and oxidative stress-related proteins in femoral arteries obtained from type 2 diabetic patients with end-stage vasculopathy. Methods Femoral arteries were obtained from 12 type 2 diabetic patients who underwent leg amputation. Segments from the femoral arteries were incubated in vitro alone and in the presence of 25 nmol l−1 FXa and 25 nmol l−1 FXa + 50 nmol l−1 rivaroxaban. Results In the femoral arteries, FXa increased triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase isotype 1 expression but decreased pyruvate dehydrogenase expression. These facts were accompanied by an increased content of acetyl-CoA. Aconitase activity was reduced in FXa-incubated femoral arteries as compared with control. Moreover, FXa increased the protein expression level of oxidative stress-related proteins which was accompanied by an increased malonyldialdehyde arterial content. The FXa inhibitor, rivaroxaban, failed to prevent the reduced expression of pyruvate dehydrogenase induced by FXa but reduced acetyl-CoA content and reverted the decreased aconitase activity observed with FXa alone. Rivaroxaban + FXa but not FXa alone increased the expression level of carnitine palmitoyltransferase I and II, two mitochondrial long chain fatty acid transporters. Rivaroxaban also prevented the increased expression of oxidative stress-related proteins induced by FXa alone. Conclusions In femoral isolated arteries from type 2 diabetic patients with end-stage vasculopathy, FXa promoted disruption of the aerobic mitochondrial metabolism. Rivaroxaban prevented such effects and even seemed to favour long chain fatty acid transport into mitochondria. PMID:25041869

  19. Acquired Factor V Inhibitor

    PubMed Central

    Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi

    2016-01-01

    Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446

  20. Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor.

    PubMed

    Petersen, L C; Bjørn, S E; Olsen, O H; Nordfang, O; Norris, F; Norris, K

    1996-01-15

    Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz- type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny , W. F., Likert, K. M., Brown, S. G., Miletich, J. R & Broze, G. J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI-(1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki = 250 nM), KPI domain 2 inhibited factor Xa (Ki = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki = 0.1 nM) and chymotrypsin (Ki = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (Ki = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three-dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki approximately 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.

  1. Coagulation factor Xa drives tumor cells into apoptosis through BH3-only protein Bim up-regulation

    SciTech Connect

    Borensztajn, Keren S. . E-mail: K.S.Borensztajn@amc.uva.nl; Bijlsma, Maarten F.; Groot, Angelique P.; Brueggemann, Lois W.; Versteeg, Henri H.; Reitsma, Pieter H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

    2007-07-15

    Coagulation Factor (F)Xa is a serine protease that plays a crucial role during blood coagulation by converting prothrombin into active thrombin. Recently, however, it emerged that besides this role in coagulation, FXa induces intracellular signaling leading to different cellular effects. Here, we show that coagulation factor (F)Xa drives tumor cells of epithelial origin, but not endothelial cells or monocytes, into apoptosis, whereas it even enhances fibroblast survival. FXa signals through the protease activated receptor (PAR)-1 to activate extracellular-signal regulated kinase (ERK) 1/2 and p38. This activation is associated with phosphorylation of the transcription factor CREB, and in tumor cells with up-regulation of the BH3-only pro-apoptotic protein Bim, leading to caspase-3 cleavage, the main hallmark of apoptosis. Transfection of tumor cells with dominant negative forms of CREB or siRNA for either PAR-1, Bim, ERK1 and/or p38 inhibited the pro-apoptotic effect of FXa. In fibroblasts, FXa-induced PAR-1 activation leads to down-regulation of Bim and pre-treatment with PAR-1 or Bim siRNA abolishes proliferation. We thus provide evidence that beyond its role in blood coagulation, FXa plays a key role in cellular processes in which Bim is the central player in determining cell survival.

  2. Factor Xa induces tissue factor expression in endothelial cells by P44/42 MAPK and NF-κB-dependent pathways

    PubMed Central

    Jiang, Rong; Wang, Ning-Ping; Tanaka, Kenichi A.; Levy, Jerrold H.; Guyton, Robert A.; Zhao, Zhi-Qing; Vinten-Johansen, Jakob

    2010-01-01

    Summary Background Tissue factor (TF) is an initiator of coagulation. The serine protease factor Xa (FXa) is the convergence point of the extrinsic and intrinsic components of the coagulation cascade. In addition to its hemostatic function, FXa elicits inflammatory responses in endothelial cells that may be important in surgical procedures in which inflammation is triggered. This study tested the hypothesis that FXa can upregulate TF on vascular endothelial cells by a MAPK- and NF-κB- dependent pathway. Methods and results Incubation of cultured human umbilical vein endothelial cells (HUVECs) with FXa increased TF protein expression and activity in a dose–dependent manner. Pre-incubation of HUVECs with the serine protease inhibitor antithrombin, which targets not only thrombin but also FXa and FIXa, inhibited FXa-induced TF expression, but the selective thrombin inhibitor hirudin did not inhibit FXa-induced TF expression, ruling out a thrombin-mediated pathway. After 10 min incubation with HUVECs, FXa rapidly induced P44/42 MAPK activation (immunoblotting of phosphorylated P44/42 MAPK) with a peak at 30 minutes. The MEK 1/2 inhibitor PD98059 partially reduced FXa-induced TF expression and activity (3.82±0.11 vs 6.54±0.08 fmol/min/cm2, P<0.05). NF-κB was activated by FXa, confirmed by cytoplasmic IkB α degradation and increased NF-κB P65 nuclear translocation. Interruption of the NF-κB pathway by the IkB α phosphorylation inhibitor Bay 11-7802 abrogated FXa-induced TF protein expression and activity (1.93± 0.02 vs 6.54±0.08 fmol/min/cm2, P<0.05). However, inhibition of PI3 kinase by LY 294002 did not attenuate FXa-induced TF protein expression and activity. Conclusions 1) FXa upregulates TF protein expression and activity in HUVECs 2) FXa-induced upregulation of TF is independent of the thrombin-PAR1 pathway, and 3) the MAPK and NF-kB pathways, but not PI3 kinase pathway, are involved in FXa-induced TF expression on human umbilical endothelial cells. FXa

  3. [Acquired coagulant factor inhibitors].

    PubMed

    Nogami, Keiji

    2015-02-01

    Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.

  4. Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

    PubMed Central

    Iyer, Janaki Krishnamurthy; Shih, Norrapat; Majumder, Munmi; Mattaparthi, Venkata Satish Kumar; Mukhopadhyay, Rupak; Doley, Robin

    2016-01-01

    In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. PMID:27089306

  5. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    PubMed Central

    Faure, Grazyna; Gowda, Veerabasappa T; Maroun, Rachid C

    2007-01-01

    Background The snake venom group IIA secreted phospholipases A2 (SVPLA2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA2-inhibitors from snake serum; and (iii) coagulation factors present in human blood. Results Using surface plasmon resonance (SPR) protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors. PMID:18062812

  6. Assessment of anti-factor Xa activity of heparin in binary parenteral nutrition admixtures for premature neonates.

    PubMed

    Foinard, A; Perez, M; Barthélémy, C; Lannoy, D; Flamein, F; Storme, L; Tournoys, A; Décaudin, B; Odou, P

    2015-07-01

    An in vitro study was carried out to determine the anti-Xa activity of heparin in binary parenteral nutrition (BPN) admixtures for premature neonates in our neonatal intensive care unit (NICU) after a 24-hour infusion, as well as to assess drug interaction with a 50% glucose solution. Two types of bags were prepared: (1) BPN admixtures (composition defined in the NICU) including sodium heparin at 77 UI/mL and (2) bags containing only G50% with sodium heparin at 193 UI/mL. The anti-Xa activity of heparin was measured in bags at T0, after the 24-hour infusion and in eluates at the outlet of the infusion line after 24hours, using a validated chromogenic anti-Xa method. Comparisons of the mean concentration observed with the theoretical value for anti-Xa activity were performed with the Student t-test. Mean values of anti-Xa activity do not differ significantly from the values expected for all conditions. We found a slight variation in anti-Xa activity when infused over 24hours for both types of bags, with and without in-line filtration, showing that heparin remains stable during this infusion period in both BPN admixtures and G50%.

  7. Independent anti-angiogenic capacities of coagulation factors X and Xa.

    PubMed

    Lange, Soledad; Gonzalez, Ibeth; Pinto, Mauricio P; Arce, Maximiliano; Valenzuela, Rodrigo; Aranda, Evelyn; Elliot, Matias; Alvarez, Marjorie; Henriquez, Soledad; Velasquez, Ethel V; Orge, Felipe; Oliva, Barbara; Gonzalez, Pamela; Villalon, Manuel; Cautivo, Kelly M; Kalergis, Alexis M; Pereira, Karla; Mendoza, Camila; Saez, Claudia; Kato, Sumie; Cuello, Mauricio A; Parborell, Fernanda; Irusta, Griselda; Palma, Veronica; Allende, Miguel L; Owen, Gareth I

    2014-11-01

    Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

  8. Isolation and study of an acquired inhibitor of human coagulation factor V.

    PubMed Central

    Nesheim, M E; Nichols, W L; Cole, T L; Houston, J G; Schenk, R B; Mann, K G; Bowie, E J

    1986-01-01

    A coagulation Factor V inhibitor developed in a man 75 yr of age in association with an anaplastic malignancy and drug treatment (including the aminoglycoside antibiotic, gentamicin). The patient did not bleed abnormally, despite both surgical challenge and plasma Factor V activity of less than 1%. The inhibited plasma had grossly prolonged prothrombin and activated partial thromboplastin times, but a normal thrombin time. Mixing studies indicated progressive coagulation inhibition with normal plasma, but not with Factor V-deficient plasma, and reversal of coagulation inhibition by the addition of bovine Factor V to the patient's plasma. 1 ml of patient plasma inhibited the Factor V activity of 90 ml of normal human plasma. The inhibitor was isolated by sequential affinity chromatography on protein A-Sepharose and Factor V-Sepharose. The IgG isolate markedly inhibits the activity of prothrombinase assembled from purified Factors Xa and Va, calcium ion, and phospholipid vesicles, and partially inhibits prothrombinase assembled from purified Factor Xa, calcium ion, and normal platelets. The Factor V of platelets, however, appears relatively inaccessible to the antibody, inasmuch as platelets isolated from whole blood supplemented for 8 h with the antibody functioned normally with respect to platelet Factor V-mediated prothrombinase function. The absence of obvious hemorrhagic difficulties in the patient, the total inhibition of plasma Factor V by the inhibitor, and the apparent inaccessibility of platelet Factor V to the inhibitor specifically implicate platelet Factor V in the maintenance of hemostasis. Images PMID:3944265

  9. Factor Xa releases matrix metalloproteinase-2 (MMP-2) from human vascular smooth muscle cells and stimulates the conversion of pro-MMP-2 to MMP-2: role of MMP-2 in factor Xa-induced DNA synthesis and matrix invasion.

    PubMed

    Rauch, Bernhard H; Bretschneider, Ellen; Braun, Marina; Schrör, Karsten

    2002-05-31

    Pro-matrix metalloproteinase-2 (pro-MMP-2) is expressed in vascular smooth muscle cells (SMCs). We report that activated coagulation factor X (FXa) induces the release of MMP-2 (65 kDa) from human SMCs. In addition, FXa cleaves pro-MMP-2 (72 kDa) into MMP-2. Pro-MMP-2 and MMP-2 were determined by gelatin zymography. MMP-2 was generated in conditioned medium containing pro-MMP-2 in a concentration-dependent fashion by FXa (3 to 100 nmol/L). FX at concentrations up to 300 nmol/L was ineffective. The conversion of pro-MMP-2 to MMP-2 was inhibited by a selective FXa inhibitor (DX-9065a) at 3 to 10 micromol/L. There was a concentration-dependent induction of an intermediate MMP-2 form (68 kDa) in lysates of FXa-treated cells. This indicates that cellular mechanisms are involved in FXa-induced conversion of pro-MMP-2. As a possible biological consequence of MMP-2 activation by FXa, DNA synthesis and matrix invasion of SMCs were determined. Both were stimulated by FXa and inhibited by the selective FXa inhibitor DX-9065a and the MMP inhibitor GM 6001 but not by hirudin or aprotinin. It is concluded that stimulation of SMCs by FXa increases the levels of MMP-2 in the extracellular space and that two different mechanisms are involved: release of active MMP-2 and cleavage of secreted pro-MMP-2. Both might contribute to the mitogenic potency of FXa and FXa-stimulated matrix invasion of SMCs.

  10. RoXaN, a Novel Cellular Protein Containing TPR, LD, and Zinc Finger Motifs, Forms a Ternary Complex with Eukaryotic Initiation Factor 4G and Rotavirus NSP3

    PubMed Central

    Vitour, Damien; Lindenbaum, Pierre; Vende, Patrice; Becker, Michelle M.; Poncet, Didier

    2004-01-01

    Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3′ consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation. PMID:15047801

  11. Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

    PubMed

    Itoh, Saotomo; Yokoyama, Ryosuke; Kamoshida, Go; Fujiwara, Toshinobu; Okada, Hiromi; Takii, Takemasa; Tsuji, Tsutomu; Fujii, Satoshi; Hashizume, Hideki; Onozaki, Kikuo

    2013-07-26

    The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

  12. Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

    PubMed Central

    Thomas, Owain; Lybeck, Emanuel; Strandberg, Karin; Tynngård, Nahreen; Schött, Ulf

    2015-01-01

    Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is

  13. The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin

    PubMed Central

    Al Otair, Hadil A.; Abdel Gader, Abdel Galil M.; Khurshid, Syed M.; Alzeer, Abdulaziz H.; Al Momen, Abdul Kareem; Al Shaikh, Mashael; Al Gahtani, Farja; Al Aseri, Zohair A.; Abdelrazik, Hossam A.H.

    2016-01-01

    Objective: Sepsis syndrome is usually accompanied by activation of blood coagulation mechanisms. Earlier studies found deficiencies of the 3 main natural anticoagulants, antithrombin, protein C, and protein S. However, none of these inhibitors block tissue factor, the prime trigger of coagulation during sepsis that is controlled specifically by the tissue factor pathway inhibitor (TFPI). The aim of this study was to characterize the fluctuations in the levels of natural anticoagulants, particularly TFPI, in the course of sepsis and to find out their association with the anticoagulant action of the low-molecular-weight heparin enoxaparin. Materials and Methods: We studied 51 consecutive patients with sepsis. Blood samples were collected from patients at baseline (0 h) and at 4, 12, and 24 h after enoxaparin administration. The following assays were undertaken using commercial kits: activated partial thromboplastin time, prothrombin time, thrombin time, total and free TFPI, protein C and protein S, antithrombin, fibrinogen, and anti-factor Xa. Results: Before enoxaparin administration, there was significant prolongation of the prothrombin time and activated partial thromboplastin time, and this remained the case in the 3 subsequent samples. There was marked reduction in the levels of antithrombin, protein C, and total and free protein S to below control values throughout the study. In contrast, plasma levels of both total and free TFPI were markedly elevated and increased after enoxaparin therapy. Anti-factor Xa levels were within the therapeutic range throughout. There was no difference in TFPI levels between those patients who died and those who survived. Conclusion: Sepsis triggered marked release of TFPI from endothelial cells. This persisted and was increased further following the administration of enoxaparin. In contrast, there was marked consumption of the natural coagulation inhibitors antithrombin, protein C, and protein S. These results go some way towards

  14. Discovery of novel heterocyclic factor VIIa inhibitors.

    PubMed

    Rai, Roopa; Kolesnikov, Aleksandr; Sprengeler, Paul A; Torkelson, Steven; Ton, Tony; Katz, Bradley A; Yu, Christine; Hendrix, John; Shrader, William D; Stephens, Robin; Cabuslay, Ronnell; Sanford, Ellen; Young, Wendy B

    2006-04-15

    Structure-activity relationships and binding mode of novel heterocyclic factor VIIa inhibitors will be described. In these inhibitors, a highly basic 5-amidinoindole moiety has been successfully replaced with a less basic 5-aminopyrrolo[3,2-b]pyridine scaffold.

  15. Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors

    SciTech Connect

    Fuchs, H.E.; Trapp, H.G.; Griffith, M.J.; Roberts, H.R.; Pizzo, S.V.

    1984-06-01

    The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the /sup 125/I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the /sup 125/I-Factor IXa was bound to ATIII by 1 min. The distribution of /sup 125/I-Factor IXa in mouse plasma was similar. The clearance of /sup 125/I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the /sup 125/I-Factor IXa was bound to ATIII. Organ distribution studies with /sup 125/I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.

  16. Improvement of low bioavailability of a novel factor Xa inhibitor through formulation of cationic additives in its oral dosage form.

    PubMed

    Fujii, Yoshimine; Kanamaru, Taro; Kikuchi, Hiroshi; Nakagami, Hiroaki; Yamashita, Shinji; Akashi, Mitsuru; Sakuma, Shinji

    2011-12-15

    A clinical trial of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naphtyl) propanoic acid (DX-9065) revealed that its oral bioavailability was only 3% when it was administered as a conventional capsule formulation. The low bioavailability of DX-9065 was likely caused by both its poor membrane permeability and its electrostatic interaction with anionic bile acids. We hypothesized that DX-9065 absorption would be enhanced when the cationic drug was free from the complex through its replacement with other cationic substances. Polystyrene nanospheres coated with cationic poly(vinylamine) and cholestyramine, which is clinically used as a cholesterol-lowering agent, dramatically prevented DX-9065 from interacting with chenodeoxycholic acid in vitro. Successive animal experiments showed that bioavailability of DX-9065 administered with these cationic substances was 2-3 times that of DX-9065 administered solely. A dry syrup formulation with one-half of a minimal cholesterol-lowering equivalent dose of cholestyramine was designed, and the clinical trial was resumed. A 1.3-fold increase in bioavailability of DX-9065 was observed when the dry syrup was administered. We successfully demonstrated that DX-9065 absorption was enhanced when the drug was administered with cationic additives; however, it appeared that the absorption-enhancing function of cholestyramine largely depended on its dose. The dose escalation is probably prerequisite for the significant improvement of DX-9065 absorption in humans.

  17. Inhibitors of the Metalloproteinase Anthrax Lethal Factor

    PubMed Central

    Goldberg, Allison B.; Turk, Benjamin E.

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LF-inhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and high-throughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection. PMID:27072692

  18. Bacithrocins A, B and C, novel thrombin inhibitors.

    PubMed

    Kamiyama, T; Umino, T; Nakamura, Y; Itezono, Y; Sawairi, S; Satoh, T; Yokose, K

    1994-09-01

    Novel thrombin inhibitors, bacithrocins A, B and C, have been isolated from the culture broth of Bacillus laterosporus Laubach NR 2988. The structures of these inhibitors have been determined to be N-acyl-L-phenylalanyl-DL-arginal by the 2D-NMR experiments on their oxidation products and by amino acid analysis. Bacithrocin A inhibits thrombin, factor Xa and trypsin with IC50s of 48, 13 and 0.65 microM, respectively, which are similar to those of bacithrocins B and C. Bacithrocins prolong the clotting time induced by thrombin and factor Xa.

  19. Activated partial thromboplastin time and anti-xa measurements in heparin monitoring: biochemical basis for discordance.

    PubMed

    Takemoto, Clifford M; Streiff, Michael B; Shermock, Kenneth M; Kraus, Peggy S; Chen, Junnan; Jani, Jayesh; Kickler, Thomas

    2013-04-01

    We examined the concordance of heparin levels measured by a chromogenic anti-Xa assay and the activated partial thromboplastin time (APTT) during unfractionated heparin therapy (UFH) and the biochemical basis for differences between these measures. We also investigated the endogenous thrombin potential (ETP) as a possible measure of anticoagulation. Paired measures of anti-Xa and APTT were performed on 569 samples from 149 patients on UFH. The anti-Xa values and the APTT were concordant in only 54% of measurements. One hundred twelve samples from 59 patients on UFH were assayed for APTT, anti-Xa, factor II, factor VIII, and ETP. Supratherapeutic APTT values but therapeutic anti-Xa results had decreased factor II activity. Subtherapeutic APTT but therapeutic anti-Xa values had high factor VIII activity. ETP correlated with anticoagulation status and UFH dose. In conclusion, factor II and VIII activity contributes to discordance between APTT and anti-Xa results. ETP measurements may provide an additional assessment of anticoagulation status.

  20. Designing Allosteric Inhibitors of Factor XIa. Lessons from the Interactions of Sulfated Pentagalloylglucopyranosides

    PubMed Central

    2015-01-01

    We recently introduced sulfated pentagalloylglucopyranoside (SPGG) as an allosteric inhibitor of factor XIa (FXIa) (Al-Horani et al., J. Med Chem.2013, 56, 867–87823316863). To better understand the SPGG–FXIa interaction, we utilized eight SPGG variants and a range of biochemical techniques. The results reveal that SPGG’s sulfation level moderately affected FXIa inhibition potency and selectivity over thrombin and factor Xa. Variation in the anomeric configuration did not affect potency. Interestingly, zymogen factor XI bound SPGG with high affinity, suggesting its possible use as an antidote. Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa. Inhibition studies in the presence of heparin showed marginal competition with highly sulfated SPGG variants but robust competition with less sulfated variants. Resolution of energetic contributions revealed that nonionic forces contribute nearly 87% of binding energy suggesting a strong possibility of specific interaction. Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa. An SPGG molecule containing approximately 10 sulfate groups on positions 2 through 6 of the pentagalloylglucopyranosyl scaffold may be the optimal FXIa inhibitor for further preclinical studies. PMID:24844380

  1. Polyphenol Compound as a Transcription Factor Inhibitor

    PubMed Central

    Park, Seyeon

    2015-01-01

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)). PMID:26529010

  2. CD-XA Reusable Launch Vehicle (RLV)

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This is the McDornell Douglas CD-XA Reusable Launch Vehicle (RLV) concept. The Delta Clipper-Experimental (DC-X) was originally developed by McDonnell Douglas for the DOD. The DC-XA is a single-stage-to-orbit, vertical takeoff/vertical landing, launch vehicle concept, whose development is geared to significantly reduce launch cost and provided a test bed for NASA Reusable Launch Vehicle (RLV) technology as the Delta Clipper-Experimental Advanced (DC-XA). The program was discontinued in 2003.

  3. High-resolution genetic mapping of rice bacterial blight resistance gene Xa23.

    PubMed

    Wang, Chunlian; Fan, Yinglun; Zheng, Chongke; Qin, Tengfei; Zhang, Xiaoping; Zhao, Kaijun

    2014-10-01

    Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating bacterial disease of rice (Oryza sativa L.), a staple food crop that feeds half of the world's population. In management of this disease, the most economical and effective approach is cultivating resistant varieties. Due to rapid change of pathogenicity in the pathogen, it is necessary to identify and characterize more host resistance genes for breeding new resistant varieties. We have previously identified the BB resistance (R) gene Xa23 that confers the broadest resistance to Xoo strains isolated from different rice-growing regions and preliminarily mapped the gene within a 1.7 cm region on the long arm of rice chromosome 11. Here, we report fine genetic mapping and in silico analysis of putative candidate genes of Xa23. Based on F2 mapping populations derived from crosses between Xa23-containing rice line CBB23 and susceptible varieties JG30 or IR24, six new STS markers Lj36, Lj46, Lj138, Lj74, A83B4, and Lj13 were developed. Linkage analysis revealed that the new markers were co-segregated with or closely linked to the Xa23 locus. Consequently, the Xa23 gene was mapped within a 0.4 cm region between markers Lj138 and A83B4, in which the co-segregating marker Lj74 was identified. The corresponding physical distance between Lj138 and A83B4 on Nipponbare genome is 49.8 kb. Six Xa23 candidate genes have been annotated, including four candidate genes encoding hypothetical proteins and the other two encoding a putative ADP-ribosylation factor protein and a putative PPR protein. These results will facilitate marker-assisted selection of Xa23 in rice breeding and molecular cloning of this valuable R gene.

  4. Mox: a novel modifier of the tomato Xa locus.

    PubMed

    Peterson, P W; Yoder, J I

    1995-01-01

    We have isolated a novel mutation that caused variegated leaf color in a tomato plant which had multiple maize Ac transposable elements and the tomato Xa allele. Xa is a previously characterized semi-dominant mutation that causes tomato leaves to be bright yellow when heterozygous (Xa/xa+). The mutation responsible for the new phenotype was named Mox (Modifier of Xa). The Mox mutation modified the Xa/xa+ yellow leaf phenotype in two ways: it compensated for the Xa allele resulting in a plant with a wildtype green color, and it caused somatic variegation which appeared as white and yellow sectors on the green background. Somatic variegation was visible only if the plant contained both the Mox and Xa loci. Genetic studies indicated that the Mox locus was linked in repulsion to Xa and that the Mox locus was genetically transmitted at a reduced frequency through the male gamete. Molecular characterization of the Ac elements in lines segregating for Mox identified an Ac insertion that appeared to cosegregate with Mox variegation. We propose a model in which the Mox mutation consists of a duplication of the xa+ allele and subsequent Ac-induced breakage of the duplicated region causes variegation.

  5. Astrocyte Mitogen Inhibitor Related to Epidermal Growth Factor Receptor

    NASA Astrophysics Data System (ADS)

    Nieto-Sampedro, Manuel

    1988-06-01

    Epidermal growth factor (EGF) is a well-characterized polypeptide hormone with diverse biological activities, including stimulation of astrocyte division. A soluble astrocyte mitogen inhibitor, immunologically related to the EGF receptor, is present in rat brain. Injury to the brain causes a time-dependent reduction in the levels of this inhibitor and the concomitant appearance of EGF receptor on the astrocyte surface. Intracerebral injection of antibody capable of binding the inhibitor caused the appearance of numerous reactive astrocytes. EGF receptor-related inhibitors may play a key role in the control of glial cell division in both normal and injured brain.

  6. Tumor necrosis factor inhibitors – state of knowledge

    PubMed Central

    Lis, Krzysztof; Kuzawińska, Olga

    2014-01-01

    Tumor necrosis factor (TNF) is considered a major proinflammatory cytokine, affecting various aspects of the immune reaction. All five TNF inhibitors currently available on the market (i.e., etanercept, infliximab, adalimumab, certolizumab and golimumab) are top sellers, although indicated only in autoimmune diseases, including rheumatoid arthritis, Crohn's disease and psoriasis. This article briefly discusses the background and place for TNF inhibitors in modern therapy. The main safety aspects of TNF inhibitor administration are described in particular, with special consideration of the available meta-analyses. Finally, perspectives on the next-generation TNF inhibitors and their use in the clinic are given. PMID:25624856

  7. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus

    PubMed Central

    Girish, Vallerinteavide Mavelli; Kini, R. Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki’ and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  8. Rituximab in the treatment of acquired factor VIII inhibitors.

    PubMed

    Wiestner, Adrian; Cho, Hearn J; Asch, Adam S; Michelis, Mary Ann; Zeller, Jack A; Peerschke, Ellinor I B; Weksler, Babette B; Schechter, Geraldine P

    2002-11-01

    Autoantibodies against factor VIII (FVIII) are rare but can cause life-threatening bleeding requiring costly factor replacement and prolonged immunosuppression. We report 4 consecutively treated patients whose acquired FVIII inhibitors responded rapidly to immunosuppressive regimens that included rituximab, a monoclonal antibody against CD20(+) B cells. Three patients had spontaneously occurring inhibitors. The fourth, a patient with mild hemophilia A, developed both an autoantibody and an alloantibody following recombinant FVIII treatment. Pretreatment FVIII activities ranged from less than 1% to 4% and inhibitor titers from 5 to 60 Bethesda units (BU). One patient with polymyalgia rheumatica who developed the inhibitor while receiving prednisone responded to single agent rituximab. The hemophilia patient had rapid resolution of the autoantibody, whereas the alloantibody persisted for months. Responses continue off treatment from more than 7 to more than 12 months. This report adds to the growing evidence that rituximab has efficacy in immune disorders resulting from autoantibody formation.

  9. Antifactor Xa levels versus activated partial thromboplastin time for monitoring unfractionated heparin.

    PubMed

    Vandiver, Jeremy W; Vondracek, Thomas G

    2012-06-01

    Intravenous unfractionated heparin (UFH) remains an important therapeutic agent, particularly in the inpatient setting, for anticoagulation. Historically, the activated partial thromboplastin time (aPTT) has been the primary laboratory test used to monitor and adjust UFH. The aPTT test has evolved since the 1950s, and the historical goal range of 1.5-2.5 times the control aPTT, which first gained favor in the 1970s, has fallen out of favor due to a high degree of variability in aPTT readings from one laboratory to another, and even from one reagent to another. As a result, it is now recommended that the aPTT goal range be based on a corresponding heparin concentration of 0.2-0.4 unit/ml by protamine titration or 0.3-0.7 unit/ml by antifactor Xa assay. Given that several biologic factors can influence the aPTT independent of the effects of UFH, many institutions have transitioned to monitoring heparin with antifactor Xa levels, rather than the aPTT. Clinical data from the last 10-20 years have begun to show that a conversion from aPTT to antifactor Xa monitoring may offer a smoother dose-response curve, such that levels remain more stable, requiring fewer blood samples and dosage adjustments. Given the minimal increased acquisition cost of the antifactor Xa reagents, it can be argued that the antifactor Xa is a cost-effective method for monitoring UFH. In this review, we discuss the relative advantages and disadvantages of the aPTT, antifactor Xa, and protamine titration tests, and provide a clinical framework to guide practitioners who are seeking to optimize UFH monitoring within their own institutions.

  10. Membranous nephropathy with acquired factor V inhibitor: a case report

    PubMed Central

    2013-01-01

    Background Membranous nephropathy is one of the most common causes of nephrotic syndrome in adults. In contrast, acquired factor V inhibitor is a rare bleeding disorder. Case presentation A 62-year-old Asian man with a history of cerebral hemorrhage, purpura, eosinophilia and hyper immunoglobulin E syndrome developed proteinuria. The bleeding disorder was diagnosed with acquired factor V inhibitors. A renal biopsy revealed that he suffered from membranous nephropathy with glomerular endothelial damage which is reported to be involved in another factor disorder. After the steroid administration, the coagulation test and proteinuria were improved. Conclusions The presence of factor V inhibitors may have been involved in the development of membranous nephropathy. PMID:24360027

  11. Anthrax lethal factor inhibitors as potential countermeasure of the infection.

    PubMed

    Kumar, B V S Suneel; Malik, Siddharth; Grandhi, Pradeep; Dayam, Raveendra; Sarma, J A R P

    2014-01-01

    Anthrax Lethal Factor (LF) is a zinc-dependent metalloprotease, one of the virulence factor of anthrax infection. Three forms of the anthrax infection have been identified: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores). Anthrax toxin is composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). Protective antigen mediates the entry of Lethal Factor/Edema Factor into the cytosol of host cells. Lethal factor (LF) inactivates mitogen-activated protein kinase kinase inducing cell death, and EF is an adenylyl cyclase impairing host defenses. In the past few years, extensive studies are undertaken to design inhibitors targeting LF. The current review focuses on the small molecule inhibitors targeting LF activity and its structure activity relationships (SAR).

  12. Discovery of Phenylglycine Lactams as Potent Neutral Factor VIIa Inhibitors.

    PubMed

    Wurtz, Nicholas R; Parkhurst, Brandon L; Jiang, Wen; DeLucca, Indawati; Zhang, Xiaojun; Ladziata, Vladimir; Cheney, Daniel L; Bozarth, Jeffrey R; Rendina, Alan R; Wei, Anzhi; Luettgen, Joseph M; Wu, Yiming; Wong, Pancras C; Seiffert, Dietmar A; Wexler, Ruth R; Priestley, E Scott

    2016-12-08

    Inhibitors of Factor VIIa (FVIIa), a serine protease in the clotting cascade, have shown strong antithrombotic efficacy in preclinical thrombosis models with minimal bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful because known chemotypes have required a highly basic group in the S1 binding pocket for high affinity. A recently reported fragment screening effort resulted in the discovery of a neutral heterocycle, 7-chloro-3,4-dihydroisoquinolin-1(2H)-one, that binds in the S1 pocket of FVIIa and can be incorporated into a phenylglycine FVIIa inhibitor. Optimization of this P1 binding group led to the first series of neutral, permeable FVIIa inhibitors with low nanomolar potency.

  13. Determination of rivaroxaban in patient’s plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS)

    PubMed Central

    Derogis, Priscilla Bento Matos; Sanches, Livia Rentas; de Aranda, Valdir Fernandes; Colombini, Marjorie Paris; Mangueira, Cristóvão Luis Pitangueira; Katz, Marcelo; Faulhaber, Adriana Caschera Leme; Mendes, Claudio Ernesto Albers; Ferreira, Carlos Eduardo dos Santos; França, Carolina Nunes; Guerra, João Carlos de Campos

    2017-01-01

    Rivaroxaban is an oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. As other new oral anticoagulants, routine monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In our study a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated to measure rivaroxaban plasmatic concentration. Our method used a simple sample preparation, protein precipitation, and a fast chromatographic run. It was developed a precise and accurate method, with a linear range from 2 to 500 ng/mL, and a lower limit of quantification of 4 pg on column. The new method was compared to a reference method (anti-factor Xa activity) and both presented a good correlation (r = 0.98, p < 0.001). In addition, we validated hemolytic, icteric or lipemic plasma samples for rivaroxaban measurement by HPLC-MS/MS without interferences. The chromogenic and HPLC-MS/MS methods were highly correlated and should be used as clinical tools for drug monitoring. The method was applied successfully in a group of 49 real-life patients, which allowed an accurate determination of rivaroxaban in peak and trough levels. PMID:28170419

  14. Anti-xa directed protocol for anticoagulation management in children supported with extracorporeal membrane oxygenation.

    PubMed

    O'Meara, L Carlisle; Alten, Jeffrey A; Goldberg, Kellen G; Timpa, Joseph G; Phillips, Jay; Laney, Debbie; Borasino, Santiago

    2015-01-01

    The optimum heparin monitoring method during extracorporeal membrane oxygenation (ECMO) is unknown. We report a protocol utilizing only anti-factor Xa (anti-Xa) to manage anticoagulation in 22 consecutive ECMO patients. Anti-Xa was monitored with heparin titration every hour until goal 0.4-0.8 IU/ml. Once therapeutic, monitoring was progressively spaced up to every 6 hours. Patients received frequent antithrombin III (ATIII). Extracorporeal membrane oxygenation indications were as follows: 13 cardiorespiratory failures, eight extracorporeal cardiopulmonary resuscitations (ECPRs), and one pulmonary hypertension. Median weight was 4 kg, age 12.5 days, and ECMO duration 88 hours. Survival was 50%. Mean heparin dose was 38 ± 11 unit/kg/hr. Eight patients received no heparin for median 9 hours because of postoperative bleeding. Compared with prior activated clotting time (ACT) protocol, there were 20 fewer blood draws per day to manage anticoagulation, p < 0.001. Only 9% of the anti-Xa levels were outside therapeutic range versus 22% using ACT, p < 0.01. Six patients had bleeding complications, and seven had oxygenator change-out. Change-out was associated with blood product administration and bleeding but not with heparin-free period (p = 0.39). Survival to discharge was higher among those who did not require circuit/oxygenator change-outs, 4/7 versus 7/7 (p < 0.01). Anti-factor Xa-based ECMO heparin management protocol is feasible, decreases blood sampling and heparin infusion adjustments, and does not appear to increase complications.

  15. Idiopathic Acquired Hemophilia A with Undetectable Factor VIII Inhibitor

    PubMed Central

    Abt, Nicholas B.; Streiff, Michael B.; Gocke, Christian B.; Kickler, Thomas S.; Lanzkron, Sophie M.

    2014-01-01

    Objective. We present the case of a 73-year-old female, with no family or personal history of a bleeding disorder, who had a classic presentation for acquired hemophilia A. Factor VIII activity was low but detectable and a factor VIII inhibitor was undetectable. Methods. The patient's plasma was comprehensively studied to determine the cause of the acquired coagulopathy. Using the Nijmegen modification of the Bethesda assay, no factor VIII autoantibody was measureable despite varying the incubation time from 1 to 3 hours. Results. The aPTT was prolonged at 46.8 seconds, which did not correct in the 4 : 1 mix but did with 1 : 1 mix. Using a one stage factor VIII activity assay, the FVIII activity was 16% and chromogenic FVIII activity was also 16%. The patient was treated with recombinant FVII and transfusion, significantly reducing bleeding. Long-term therapy was initiated with cyclophosphamide and prednisone with normalization of FVIII activity. Conclusions. Physicians can be presented with the challenging clinical picture of an acquired factor VIII inhibitor without a detectable inhibitor by the Bethesda assay. Standard therapy for an acquired hemophilia A should be considered. PMID:24955264

  16. A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

    PubMed

    Bai, Fengwei; Asojo, Oluwatoyin A; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A; Leng, Lin; Koski, Raymond A; Powell, Thomas J; Bucala, Richard; Anthony, Karen G

    2012-08-31

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

  17. Factor XIa inhibitors: A review of patent literature

    PubMed Central

    Al-Horani, Rami A.; Desai, Umesh R.

    2016-01-01

    Introduction Anticoagulants are the mainstay for prevention and/or treatment of thrombotic disorders. Each clinically used anticoagulant is associated with significant adverse consequences, especially bleeding. Factor XIa (FXIa), a key factor involved in the amplification of procoagulation signal, has been suggested as a major target for anticoagulant drug discovery because of reduced risk of bleeding. Areas covered Our literature search uncovered dozens of industrial and academic patents on the discovery of novel FXIa/FXI inhibitors. Small peptidomimetics, sulfated glycosaminoglycan mimetics, polypeptides, antisense oligonucleotides, and monoclonal antibodies have been developed as inhibitors of FXIa. Although many agents are in early discovery/development phases, the activity and safety of a few have been evaluated in various animal models and in humans. Expert opinion FXIa is a promising drug target for development of effective anticoagulants with limited bleeding complications. Literature reveals a major trend in the number of patent applications over the last three years. These inhibitors exploit different approaches for target inhibition. Allosteric modulation of FXIa and biosynthetic inhibition of FXI are mechanistically unique. Despite initial results in patients undergoing knee anthroplasty as with antisense oligonucleotides, major advances should be realized, particularly with respect to pharmacokinetics, for FXI/FXIa inhibitors to enter the clinic. PMID:26881476

  18. International biological standards for coagulation factors and inhibitors.

    PubMed

    Hubbard, Anthony R

    2007-04-01

    The use of international biological standards during the last 30 years has proved extremely successful in promoting global harmonization of estimates between laboratories and methods. Experience has led to the identification of physical criteria essential for standards to be suitable for long-term use. High precision of liquid filling coupled with low residual moisture and oxygen and the use of sealed glass ampoules have been found consistent with homogeneous and stable International Standards (ISs). Most plasma coagulation factors and inhibitors are calibrated in International Units (IU), which are defined as the amount of analyte in 1 mL of normal pooled plasma. Adoption of the IU has provided clarity in the definition of normal and abnormal states and has facilitated dose calculation for replacement therapy. The assay of like-versus-like materials (e.g., concentrate versus concentrate) has been found to improve interlaboratory agreement and there are now both plasma and concentrate ISs available for many coagulation factors and inhibitors. Studies into the assay of recombinant factor VIII have indicated that additional measures, such as modifications to assay methodology, are necessary to reduce interlaboratory variability. This experience may prove valuable in the future, when we have to deal increasingly with the challenges to standardization associated with the products of bioengineering.

  19. Draft Genome Sequence of Enterobacter cloacae subsp. cloacae Strain 08XA1, a Fecal Bacterium of Giant Pandas

    PubMed Central

    Yan, Yue; Zhao, Chuan-Wu; Zhang, Yi-Zheng; Zhang, Zhi-He; Pan, Guang-Lin; Liu, Wen-Wang; Ma, Qing-Yi; Hou, Rong

    2012-01-01

    Enterobacter cloacae, a common pathogenic bacterium, is a Gram-negative bacillus. We analyzed the draft genome of Enterobacter cloacae subsp. cloacae strain 08XA1 from the feces of a giant panda in China. Genes encoding a β-lactamase and efflux pumps, as well as other factors, have been found in the genome. PMID:23209197

  20. New insights into the biology of tissue factor pathway inhibitor

    PubMed Central

    MARONEY, S. A.; MAST, A. E.

    2015-01-01

    Summary Tissue factor pathway inhibitor (TFPI) dampens the initiation of blood coagulation by inhibiting two potent procoagulant complexes, tissue factor–factor VIIa (TF–FVIIa) and early forms of prothrombinase. TFPI isoforms, TFPIα and TFPIβ, result from alternative splicing of mRNA, producing distinct C-terminal ends of the two proteins. Both isoforms inhibit TF–FVIIa, but only TFPIα can inhibit early forms of prothrombinase by binding of its positively charged C-terminus with high affinity to the acidic B-domain exosite of FVa, which is generated upon activation by FXa. TFPIα and TFPIβ are produced in cultured human endothelial cells, while platelets contain only TFPIα. Knowledge of the anticoagulant mechanisms and tissue expression patterns of TFPIα and TFPIβ have improved our understanding of the phenotypes observed in different mouse models of TFPI deficiency, the east Texas bleeding disorder, and the development of pharmaceutical agents that block TFPI function to treat hemophilia. PMID:26149025

  1. Treatment of rheumatoid arthritis with tumour necrosis factor inhibitors

    PubMed Central

    Mewar, Devesh; Wilson, Anthony G

    2011-01-01

    Advances in our understanding of the key mediators of chronic inflammation and tissue damage characteristic of rheumatoid arthritis (RA) have resulted in the development of novel therapies primarily targeting pro-inflammatory cytokines. Inhibitors of tumour necrosis factor (TNF) are the most widely used of the biological therapies at present with five different agents currently available; four are based on monoclonal anti-TNF antibodies and a soluble TNF receptor-Fc fusion protein. Long-term use of these molecules has proven to be highly effective in the majority of patients; however, around one-third have a suboptimal response potentially leading to further cartilage and bone damage, furthermore these agents are expensive compared with conventional therapies such as methotrexate. Many recent studies have attempted to identify therapeutic response biomarkers of TNF inhibitors which could be used to improve therapeutic targeting. The presence of rheumatoid factor and anti-cyclic citullinated protein antibodies, present in around 65% of RA patients, are associated with a poorer response to anti-TNF agents. Poorer response is also associated with levels of C-reactive protein and cartilage degradation product at initiation of treatment. Intriguingly, genetic studies of variants of TNF and of genes encoding members of the Toll-like receptors, nuclear factor-kappa B and p38 mitogen-activated protein kinase signalling families have been associated with response to individual anti-TNF agents. Continued advances in technologies such as ultra high throughput sequencing and proteomics should facilitate the discovery of additional biomarkers of response to anti-TNF resulting in improved disease control and quality of life for RA patients and reduced costs for healthcare funders. PMID:21039421

  2. Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21.

    PubMed

    Ellur, Ranjith K; Khanna, Apurva; S, Gopala Krishnan; Bhowmick, Prolay K; Vinod, K K; Nagarajan, M; Mondal, Kalyan K; Singh, Nagendra K; Singh, Kuldeep; Prabhu, Kumble Vinod; Singh, Ashok K

    2016-07-11

    Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India's forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region.

  3. Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21

    PubMed Central

    Ellur, Ranjith K.; Khanna, Apurva; S, Gopala Krishnan.; Bhowmick, Prolay K.; Vinod, K. K.; Nagarajan, M.; Mondal, Kalyan K.; Singh, Nagendra K.; Singh, Kuldeep; Prabhu, Kumble Vinod; Singh, Ashok K.

    2016-01-01

    Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India’s forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region. PMID:27403778

  4. Therapeutic inhibitors of tumor necrosis factor in Crohn's disease.

    PubMed

    Ganesan, Srinivasan; Travis, Simon P L; Ahmad, Tariq; Jazrawi, Riadh

    2002-09-01

    Therapeutic options for patients with refractory ulcerative colitis or Crohn's disease have recently been augmented by the introduction of biological therapies. The pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha is present in elevated concentrations in patients with inflammatory bowel disease and inhibitors of TNF alpha have proved effective as treatment. Strategies aimed at reducing TNF in patients with Crohn's disease, include the mouse/human chimeric monoclonal antibody, infliximab (Centocor Inc), the humanized monoclonal antibody, CDP-571 (Celltech Group plc), the human recombinant TNF receptor fusion protein, etanercept (Immunex Corp), and thalidomide. New approaches, including the use of soluble TNF receptors, appear promising. This article reviews the evidence of therapeutic inhibition of TNF.

  5. A comparison of red blood cell transfusion utilization between anti-activated factor X and activated partial thromboplastin monitoring in patients receiving unfractionated heparin.

    PubMed

    Belk, K W; Laposata, M; Craver, C

    2016-11-01

    Essentials Anti-activated factor X (Anti-Xa) monitoring is more precise than activated partial thromboplastin (aPTT). 20 804 hospitalized cardiovascular patients monitored with Anti-Xa or aPTT were analyzed. Adjusted transfusion rates were significantly lower for patients monitored with Anti-Xa. Adoption of Anti-Xa protocols could reduce transfusions among cardiovascular patients in the US.

  6. Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway-induced resistance to vascular endothelial growth factor receptor inhibitor.

    PubMed

    Nakagawa, Takayuki; Matsushima, Tomohiro; Kawano, Satoshi; Nakazawa, Youya; Kato, Yu; Adachi, Yusuke; Abe, Takanori; Semba, Taro; Yokoi, Akira; Matsui, Junji; Tsuruoka, Akihiko; Funahashi, Yasuhiro

    2014-06-01

    Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

  7. Development of the epidermal growth factor receptor inhibitor OSI-774.

    PubMed

    Grünwald, Viktor; Hidalgo, Manuel

    2003-06-01

    The epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in the regulation of a complex array of essential biological processes such as cell proliferation and survival. Dysregulation of the EGFR signaling network has been frequently reported in multiple human cancers and has been associated with the processes of tumor development, growth, proliferation, metastasis, and angiogenesis. Inhibition of the EGFR was associated with antitumor effects in preclinical models. On the basis of these data, therapeutics targeting the EGFR were explored in clinical trials. OSI-774 is a small-molecule selective inhibitor of the EGFR tyrosine kinase. In preclinical studies, OSI-774 inhibited the phosphorylation of the EGFR in a dose-dependent and concentration-dependent manner resulting in cell cycle arrest and induction of apoptosis. In in vivo studies, this agent caused tumor growth inhibition and showed synergistic effects when combined with conventional chemotherapy. Subsequent single-agent phase I studies and phase I studies in combination with chemotherapy showed that the agent has a good safety profile and induced tumor growth inhibition in a substantial number of patients with a variety of different solid tumors. Preliminary reports from phase II studies confirmed the excellent tolerability of OSI-774 and showed encouraging preliminary activity. Phase III studies have either been completed or are ongoing in several tumor types such as lung cancer and pancreatic cancer. In summary, OSI-774 is a novel inhibitor of the EGFR tyrosine kinase that has shown promising activity in initial studies and is currently undergoing full development as an anticancer drug.

  8. Cell penetrating peptide inhibitors of Nuclear Factor-kappa B

    PubMed Central

    Orange, J. S.; May, M. J.

    2010-01-01

    The nuclear factor kappa B (NF-κB) transcription factors are activated by a range of stimuli including pro-inflammatory cytokines. Active NF-κB regulates the expression of genes involved in inflammation and cell survival and aberrant NF-κB activity plays pathological roles in certain types of cancer and diseases characterized by chronic inflammation. NF-κB signaling is an attractive target for the development of novel anti-inflammatory or anti-cancer drugs and we discuss here how the method of peptide transduction has been used to specifically target NF-κB. Peptide transduction relies on the ability of certain small cell-penetrating peptides (CPPs) to enter cells, and a panel of CPP-linked inhibitors (CPP-Is) has been developed to directly inhibit NF-κB signaling. Remarkably, several of these NF-κB-targeting CPP-Is are effective in vivo and therefore offer exciting potential in the clinical setting. PMID:18668204

  9. Induction of Xa10-like genes in rice cultivar Nipponbare confers disease resistance to rice bacterial blight.

    PubMed

    Wang, Jun; Tian, Dongsheng; Gu, Keyu; Yang, Xiaobei; Wang, Lanlan; Zeng, Xuan; Yin, Zhongchao

    2017-03-17

    Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive bacterial diseases throughout the major rice growing regions in the world. The rice disease resistance (R) genes Xa10 confers race-specific disease resistance to X. oryzae pv. oryzae strains that deliver the corresponding transcription activator-like (TAL) effectors AvrXa10. Upon bacterial infection, AvrXa10 binds specifically to the effector binding element (EBE) in the promoter of the R gene and activates its expression. Xa10 encodes an executor R protein that triggers hypersensitive response and activates disease resistance. Rice cultivar Nipponbare carries two Xa10-like genes in its genome, of which one is the susceptible allele of the Xa23 gene, a Xa10-like TAL effector-dependent executor R gene isolated recently from rice cultivar CBB23. However, the function of the two Xa10-like genes in disease resistance to X. oryzae pv. oryzae strains has not been investigated. Here we designated the two Xa10-like genes as Xa10-Ni and Xa23-Ni and characterized their function for disease resistance to rice bacterial blight. Both Xa10-Ni and Xa23-Ni provided disease resistance to X. oryzae pv. oryzae strains that deliver the matching artificially designed TAL effectors (dTALEs). Transgenic rice plants containing Xa10-Ni and Xa23-Ni under the Xa10 promoter provided specific disease resistance to X. oryzae pv. oryzae strains that deliver AvrXa10. Xa10-Ni and Xa23-Ni knock-out mutants abolished dTALE-dependent disease resistance to X. oryzae pv. oryzae. Heterologous expression of Xa10-Ni and Xa23-Ni in Nicotiana benthamiana triggered cell death. The 19-amino acid residues at the N-terminal regions of XA10 or XA10-Ni are dispensable for their function in inducing cell death in N. benthamiana and the C-terminal regions of XA10, XA10-Ni and XA23-Ni are interchangeable among each other without affecting their function. Like XA10, both XA10-Ni and XA23-Ni locate to the endoplasmic

  10. Identification of factor inhibitors by diagnostic haemostasis laboratories: a large multi-centre evaluation.

    PubMed

    Favaloro, Emmanuel J; Bonar, Roslyn; Duncan, Elizabeth; Earl, Gail; Low, Joyce; Aboud, Margaret; Just, Sarah; Sioufi, John; Street, Alison; Marsden, Katherine

    2006-07-01

    We have assessed the proficiency of diagnostic haemostasis facilities to correctly identify coagulation factor abnormalities and inhibitors. Forty-two laboratories participating in the external Quality Assurance Program (QAP) conducted by the RCPA agreed to participate and were each sent a set of eight samples (each 3 x 1 ml) for evaluation. They were asked to blind test these samples for the presence or absence of inhibitors, and where identified, to perform further analysis (including specific inhibitor analysis). In order to make the exercise more challenging, in addition to true factor inhibitors, samples were provided that reflected potential pre-analytical variables that might arise and complicate inhibitor detection or lead to false inhibitor identification. In brief, the sample set comprised a true high level factor (F) V inhibitor, a true moderate level FVIII inhibitor (but sample was defibrinogenated), a true lupus anticoagulant (LA), a normal (but slightly aged) plasma sample, a normal serum sample, a normal EDTA sample, an oral anticoagulant/vitamin K deficiency sample, and a gross heparin ( approximately 10 U/ml) contaminated sample. Sixty-three percent of participants correctly identified the true FV inhibitor as such, although the reported range varied greatly [10 to >250 Bethesda units (BU/ml)] and 46% correctly identified the true FVIII inhibitor, despite the complication of the sample presentation, although the reported range also varied (7 to 64 BU/ml). Some laboratories either failed to identify the inhibitor present, or misidentified the inhibitor type. The LA, the oral anticoagulant/vitamin K deficiency, the normal serum sample, and the normal (aged) sample were also correctly identified by most laboratories, as was the absence of specific factor inhibitors in these samples. However, a small subset of laboratories incorrectly identified the presence of specific factor inhibitors in some of these samples. The heparin sample was also correctly

  11. Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

    PubMed Central

    Walsh, P N; Sinha, D; Kueppers, F; Seaman, F S; Blankstein, K B

    1987-01-01

    We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug. Images PMID:3500185

  12. Synthesis and P1' SAR exploration of potent macrocyclic tissue factor-factor VIIa inhibitors

    SciTech Connect

    Ladziata, Vladimir; Glunz, Peter W.; Zou, Yan; Zhang, Xiaojun; Jiang, Wen; Jacutin-Porte, Swanee; Cheney, Daniel L.; Wei, Anzhi; Luettgen, Joseph M.; Harper, Timothy M.; Wong, Pancras C.; Seiffert, Dietmar; Wexler, Ruth R.; Priestley, E. Scott

    2016-10-01

    Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1'-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1' group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1' substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

  13. [New anticoagulants - direct thrombin inhibitors].

    PubMed

    Brand, B; Graf, L

    2012-11-01

    Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.

  14. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    PubMed

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols.

  15. Genetic factors influencing inhibitor development in a cohort of South African haemophilia A patients.

    PubMed

    Lochan, A; Macaulay, S; Chen, W C; Mahlangu, J N; Krause, A

    2014-09-01

    A critical complication of factor VIII (FVIII) replacement therapy in Haemophilia A (HA) treatment is inhibitor development. Known genetic factors predisposing to inhibitor development include FVIII (F8) gene mutations, ethnicity, a family history of inhibitors and FVIII haplotype mismatch. The aim of this study was to characterize and correlate these genetic factors in a cohort of South African HA patients. This was a retrospective study that included 229 patients and involved the analysis of patient files, HA molecular and clinical databases and molecular analysis of the F8 gene haplotype. Of the 229 patients, 51% were of black ethnicity, 49% were white, 5% had mild HA, 4% were moderate and 91% were severe, 36% were int22 positive and 13% were inhibitor positive. Of the inhibitor positive patients, 72% were black patients. Inhibitors were reported in 27% of black int22 positive patients, 13% of black int22 negative patients, 9% of white int22 positive patients and 7% of white int22 negative. The H1 haplotype was more common in whites (75%) and H2 was more common in blacks (74%). H3 and H5 were only found in black patients and had a higher frequency of inhibitor development than H1 and H2. In this small HA cohort, black patients had a significantly higher frequency of inhibitor development and the results were indicative of an association between inhibitor development, ethnicity and haplotype.

  16. Laboratory identification of factor VIII inhibitors in the real world: the experience from Australasia.

    PubMed

    Favaloro, E J; Bonar, R; Kershaw, G; Mohammed, S; Duncan, E; Marsden, K

    2010-07-01

    The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titres during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either using the original or Nijmegen-modified Bethesda assay. In view of previously demonstrated high variability in laboratory results for inhibitor assays, we have more extensively examined laboratory performance in the identification of FVIII inhibitors. Over the past 3 years, we conducted two questionnaire-based surveys and two wet-challenge surveys utilizing eight samples comprising no FVIII inhibitor (n = 1), or low-titre (n = 2), medium-titre (n = 3) or high-titre (n = 2) FVIII inhibitor. Four samples were tested by 42 laboratories in 2007, and four by 52 laboratories in 2009. High inter-laboratory variation was evident, with CVs around 50% not uncommon, and some 10% of all laboratories (or around 15% of laboratories using Bethesda method) failed to detect low-level inhibitors of around 1 BU mL(-1). Laboratories using the Nijmegen method appeared to perform better than those using a standard Bethesda assay, with lower evident assay variation and no false negatives. There was a wide variety of laboratory practice, with no two laboratories using exactly the same process for testing and interpretation of factor inhibitor findings. In conclusion, our study indicates that there is still much need for standardization and improvement in factor inhibitor detection, and we hope that our findings provide a basis for future improvements in this area.

  17. The prevalence of factor VIII and IX inhibitors among Saudi patients with hemophilia

    PubMed Central

    Owaidah, Tarek; Momen, Abdulkareem Al; Alzahrani, Hazzaa; Almusa, Abdulrahman; Alkasim, Fawaz; Tarawah, Ahmed; Nouno, Randa Al; Batniji, Fatima Al; Alothman, Fahad; Alomari, Ali; Abu-Herbish, Saud; Abu-Riash, Mahmoud; Siddiqui, Khawar; Ahmed, Mansor; Mohamed, SY; Saleh, Mahasen

    2017-01-01

    Abstract Hemophilia A and B are X-linked diseases that predominantly affect male patients. Patients can develop coagulation factor inhibitors, which exponentially increases the treatment cost. However, the prevalence of factor VIII and IX inhibitors in Saudi Arabia is unclear. This study aimed to determine the Saudi prevalence of factor VIII and IX inhibitors. This 4-year, 7-center, cross-sectional study evaluated the Saudi prevalences of hemophilia A and B. We collected the patients’ clinical data, evaluated their disease, and tested for factor inhibitors. We included 202 patients with hemophilia (median age at diagnosis: 0.13 years, range: birth–34.8 years). The patients included 198 male patients (98%), 148 patients with hemophilia A (73.3%), and 54 patients with hemophilia B (26.7%). The patients exhibited severe factor VIII activity (<1%; 121 patients; 5.2%), moderate activity (1–5%; 7 patients; 4.9%), and mild activity (14 patients; 9.9%). Among the patients with care-related data, most patients were treated for episodic bleeding (76.8%) or received prophylaxis (22.6%); 1 patient received both treatments. Among the patients with source-related data, the factor replacements were derived from plasma (48.4%), recombinant concentrates (22.9%), both sources (14.6%), or fresh frozen plasma (14.1%). Factor VIII inhibitors were observed in 43 (29.3%) of the 147 patients, and only 1 of the 54 patients developed factor IX inhibitors. Most patients who developed inhibitors had severe hemophilia (40/44; 90.9%), and inhibitors were also common among patients who received recombinant products (14/43; 32.6%). The Saudi prevalence of factor inhibitors was similar to those among other ethnic populations. PMID:28079788

  18. Treatment of patients with hemophilia A and inhibitors to factor FVIII with cimetidine.

    PubMed

    Ambriz Fernandez, R; Quintana Gonzalez, S; Martinez Murillo, C; Dominguez Garcia, V; Rodriguez Moyado, H; Collazo Jaloma, J

    1996-01-01

    In this study, cimetidine was used to treat patients with hemophilia A and inhibitors to factor VIII who presented with acute hemorrhages (Group A) and those without hemorrhages (Group B). The dose of cimetidine was 15 mg/kg/day. Group A consisted of five patients with inhibitors between 156 and > 10,000 Bethesda Units (BU), all with serious hemorrhagic problems. The control of hemorrhaging was effective in 100% of these patients, although inhibitor levels remained high (25-380 BU). Group B consisted of seven patients who did not have hemorrhages, whose inhibitor levels were 41-358 BU. Five of these patients no longer had anamnestic responses to Factor VIII after several months of treatment with cimetidine. No difference in the response to cimetidine was seen between HIV positive and HIV negative patients. The results suggest that cimetidine is useful to suppress inhibitors to Factor VIII in patients with hemophilia A.

  19. Protease inhibitors interfere with the necessary factors of carcinogenesis.

    PubMed

    Troll, W

    1989-05-01

    Many tumor promoters are inflammatory agents that stimulate the formation of oxygen radicals (.O2-) and hydrogen peroxide (H2O2) in phagocytic neutrophils. The neutrophils use the oxygen radicals to kill bacteria, which are recognized by the cell membrane of phagocytic cells causing a signal to mount the oxygen response. The tumor promoter isolated from croton oil, 12-O-tetradecanoylphorbol-13-acetate (TPA), mimics the signal, causing an oxygen radical release that is intended to kill bacteria; instead, it injures cells in the host. Oxygen radicals cause single strand breaks in DNA and modify DNA bases. These damaging reactions appear to be related to tumor promotion, as three types of chemopreventive agents, retinoids, onion oil, and protease inhibitors, suppress the induction of oxygen radicals in phagocytic neutrophils and suppress tumor promotion in skin cancer in mice. Protease inhibitors also suppress breast and colon cancers in mice. Protease inhibitors capable of inhibiting chymotrypsin show a greater suppression of the oxygen effect and are better suppressors of tumor promotion. In addition, oxygen radicals may be one of the many agents that cause activation of oncogenes. Since retinoids and protease inhibitors suppress the expression of the ras oncogene in NIH 3T3 cells, NIH 3T3 cells may serve as a relatively facile model for finding and measuring chemopreventive agents that interfere with the carcinogenic process.

  20. XA21-specific induction of stress-related genes following Xanthomonas infection of detached rice leaves

    PubMed Central

    Liu, Furong; Chen, Huamin; Wei, Tong; Nguyen, Yen P.; Shaker, Isaac W.F.

    2016-01-01

    The rice XA21 receptor kinase confers robust resistance to the bacterial pathogen Xanthomonas oryzaepv. oryzae (Xoo). We developed a detached leaf infection assay to quickly and reliably measure activation of the XA21-mediated immune response using genetic markers. We used RNA sequencing of elf18 treated EFR:XA21:GFP plants to identify candidate genes that could serve as markers for XA21 activation. From this analysis, we identified eight genes that are up-regulated in both in elf18 treated EFR:XA21:GFP rice leaves and Xoo infected XA21 rice leaves. These results provide a rapid and reliable method to assess bacterial-rice interactions. PMID:27703843

  1. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

    PubMed Central

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R.; Kehn-Hall, Kylene; Omichinski, James G.

    2015-01-01

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  2. Pattern of factor VIII inhibitors in patients with hemophilia A in the north east of Iran.

    PubMed

    Modaresi, A R; Torghabeh, H Mansouri; Pourfathollah, A A; Shooshtari, M Mahmoodian; Yazdi, Z Rezaie

    2006-06-01

    This survey was conducted to evaluate coagulation factor VIII:C inhibitors among 102 hemophilia A patients from different cities of Khorasan province in north east of Iran in order to identify and characterize the pattern of inhibitor formation in these patients population. For this purpose, we randomly obtained plasma samples of 102 hemophilia A patients (44 patients with severe, 28 patients with intermediate and 30 patients with mild hemophilia A) and studied them using two tests: the APTT mix and Bethesda test were performed. In the whole group 20 patients (19.6%) factor VIII inhibitors were detected. These were in 11 patients with severe, five patients with intermediate and four patients with mild hemophilia A. None of patients with hemophilia A had previously been studied for the presence of an inhibitor, so there was no existing history of inhibitor evaluation.

  3. Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection.

    PubMed

    Peng, Hai; Chen, Zheng; Fang, Zhiwei; Zhou, Junfei; Xia, Zhihui; Gao, Lifen; Chen, Lihong; Li, Lili; Li, Tiantian; Zhai, Wenxue; Zhang, Weixiong

    2015-07-17

    Rice bacterial blight (BB) is a devastating rice disease. The Xa21 gene confers a broad and persistent resistance against BB. We introduced Xa21 into Oryza sativa L ssp indica (rice 9311), through multi-generation backcrossing, and generated a nearly isogenic, blight-resistant 9311/Xa21 rice. Using next-generation sequencing, we profiled the transcriptomes of both varieties before and within four days after infection of bacterium Xanthomonas oryzae pv. oryzae. The identified differentially expressed (DE) genes and signaling pathways revealed insights into the functions of Xa21. Surprisingly, before infection 1,889 genes on 135 of the 316 signaling pathways were DE between the 9311/Xa21 and 9311 plants. These Xa21-mediated basal pathways included mainly those related to the basic material and energy metabolisms and many related to phytohormones such as cytokinin, suggesting that Xa21 triggered redistribution of energy, phytohormones and resources among essential cellular activities before invasion. Counter-intuitively, after infection, the DE genes between the two plants were only one third of that before the infection; other than a few stress-related pathways, the affected pathways after infection constituted a small subset of the Xa21-mediated basal pathways. These results suggested that Xa21 primed critically important genes and signaling pathways, enhancing its resistance against bacterial infection.

  4. A Spider-Derived Kunitz-Type Serine Protease Inhibitor That Acts as a Plasmin Inhibitor and an Elastase Inhibitor

    PubMed Central

    Wan, Hu; Lee, Kwang Sik; Kim, Bo Yeon; Zou, Feng Ming; Yoon, Hyung Joo; Je, Yeon Ho; Li, Jianhong; Jin, Byung Rae

    2013-01-01

    Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K+ channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (Ki 7.34 nM) and chymotrypsin (Ki 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (Ki 4.89 nM) and neutrophil elastase (Ki 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor. PMID:23308198

  5. Sulfated Pentagalloylglucoside is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

    PubMed Central

    Al-Horani, Rami A.; Ponnusamy, Pooja; Mehta, Akul Y.; Gailani, David; Desai, Umesh R.

    2013-01-01

    Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced VMAX of FXIa hydrolysis of chromogenic substrate without affecting the KM suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance. PMID:23316863

  6. What factors determine patients' preference for tumour necrosis factor inhibitors in ankylosing spondylitis?

    PubMed

    Fajri, Dessy W; Brand, Caroline A; Dharmage, Shyamali C; Martin, Belinda J; Buchanan, Russell R C; Schachna, Lionel

    2009-05-01

    Tumour necrosis factor inhibitor (TNFi) therapy, either intravenous (IV) or subcutaneous (SQ), demonstrates similar efficacy in ankylosing spondylitis (AS). The objective of this study was to examine factors influencing patient preference of TNFi. Fifty-nine (79.7%) participants were male with mean age 43.9 years and disease duration of 22.0 years. Fifty-nine patients (79.7%) agreed with the statement 'My doctor gave me a choice and I made a decision based on my personal preference'. Patients commenced first on IV TNFi most commonly cited reduced frequency of injections (96.6%), administration by a trained professional (89.7%) and use of infusion time for leisure activities (86.2%). Patients commenced on SQ TNFi cited flexibility with timing of treatment (80%), shortened administration time (73.3%) and the convenience of home therapy (73.3%). Shared clinical decision-making between clinicians and patients may be desirable for AS patients commencing TNFi therapy.

  7. Acquired factor V inhibitor after exposure to topical human thrombin related to an otorhinolaryngological procedure.

    PubMed

    Donohoe, K; Levine, R

    2015-10-01

    Acquired factor V (FV) inhibitors occur rarely and classically develop after exposure to bovine thrombin. The clinical presentation is variable, ranging from asymptomatic with incidental laboratory abnormalities to significant bleeding. With the development of human-derived thrombin agents, bovine thrombin is less frequently used. We report a case of an acquired FV inhibitor that developed in a patient after exposure to human thrombin used as a hemostatic agent during an otorhinolaryngology surgical procedure. Our review of the literature revealed only one prior reported case of FV inhibitor after exposure to human thrombin. Hematologists and surgeons should be aware of this rare, but potentially life-threatening, complication in the postprocedural setting.

  8. Bleeding associated with acquired factor V inhibitor in a patient on warfarin treated successfully with prednisolone

    PubMed Central

    Khalafallah, Alhossain; Grabek, Julian; Hayes, Robert; Mohamed, Muhajir

    2013-01-01

    An 85-year-old man on warfarin for atrial fibrillation presented with skin bleeding. International normalised ratio (INR) and activated partial thromboplastin time (APTT) were elevated and did not correct even after warfarin reversal with vitamin K, prothrombin complex concentrate (PCC) and fresh frozen plasma. Mixing coagulation studies with normal plasma suggested the presence of an inhibitor rather than the multiple coagulation factor deficiencies expected with warfarin. Assays of the common-pathway coagulation factors revealed factor V concentration <2% with inhibitor level elevated to 11 Bethesda units. The bleeding resolved following a course of corticosteroids. Coagulation studies and factor V level returned to normal along with resolution of the inhibitor. We report the case of the diagnostic dilemma posed and successful therapy implemented despite the limited evidence-based data being available for the treatment of this rare condition. PMID:23921688

  9. Factor VIII gene (F8) mutation and risk of inhibitor development in nonsevere hemophilia A.

    PubMed

    Eckhardt, Corien L; van Velzen, Alice S; Peters, Marjolein; Astermark, Jan; Brons, Paul P; Castaman, Giancarlo; Cnossen, Marjon H; Dors, Natasja; Escuriola-Ettingshausen, Carmen; Hamulyak, Karly; Hart, Daniel P; Hay, Charles R M; Haya, Saturnino; van Heerde, Waander L; Hermans, Cedric; Holmström, Margareta; Jimenez-Yuste, Victor; Keenan, Russell D; Klamroth, Robert; Laros-van Gorkom, Britta A P; Leebeek, Frank W G; Liesner, Ri; Mäkipernaa, Anne; Male, Christoph; Mauser-Bunschoten, Evelien; Mazzucconi, Maria G; McRae, Simon; Meijer, Karina; Mitchell, Michael; Morfini, Massimo; Nijziel, Marten; Oldenburg, Johannes; Peerlinck, Kathelijne; Petrini, Pia; Platokouki, Helena; Reitter-Pfoertner, Sylvia E; Santagostino, Elena; Schinco, Piercarla; Smiers, Frans J; Siegmund, Berthold; Tagliaferri, Annarita; Yee, Thynn T; Kamphuisen, Pieter Willem; van der Bom, Johanna G; Fijnvandraat, Karin

    2013-09-12

    Neutralizing antibodies (inhibitors) toward factor VIII form a severe complication in nonsevere hemophilia A, profoundly aggravating the bleeding pattern. Identification of high-risk patients is hampered by lack of data that take exposure days to therapeutic factor VIII concentrates into account. In the INSIGHT study, we analyzed the association between F8 mutation and inhibitor development in patients with nonsevere hemophilia A (factor VIII 2-40 IU/dL). This analysis included 1112 nonsevere hemophilia A patients from 14 centers in Europe and Australia that had genotyped at least 70% of their patients. Inhibitor risk was calculated as Kaplan-Meier incidence with cumulative number of exposure days as the time variable. During 44 800 exposure days (median, 24 exposure days per patient; interquartile range [IQR], 7-90), 59 of the 1112 patients developed an inhibitor; cumulative incidence of 5.3% (95% confidence interval [CI], 4.0-6.6) after a median of 28 exposure days (IQR, 12-71). The inhibitor risk at 50 exposure days was 6.7% (95% CI, 4.5-8.9) and at 100 exposure days the risk further increased to 13.3% (95% CI, 9.6-17.0). Among a total of 214 different F8 missense mutations 19 were associated with inhibitor development. These results emphasize the importance of F8 genotyping in nonsevere hemophilia A.

  10. Effect of food, an antacid, and the H2 antagonist ranitidine on the absorption of BAY 59-7939 (rivaroxaban), an oral, direct factor Xa inhibitor, in healthy subjects.

    PubMed

    Kubitza, Dagmar; Becka, Michael; Zuehlsdorf, Michael; Mueck, Wolfgang

    2006-05-01

    To investigate the influence of food and administration of an antacid (aluminum-magnesium hydroxide) or ranitidine on the absorption of BAY 59-7939 (rivaroxaban), 4 randomized studies were performed in healthy male subjects. In 2 food interaction studies, subjects received BAY 59-7939, either as two 5-mg tablets (fasted and fed), four 5-mg tablets (fasted), or one 20-mg tablet (fasted and fed). In 2 drug interaction studies, BAY 59-7939 (six 5-mg tablets) was given alone or with ranitidine (150 mg twice daily, preceded by a 3-day pretreatment phase) or antacid (10 mL). Plasma samples were obtained to assess pharmacokinetic and pharmacodynamic parameters of BAY 59-7939. In the presence of food, time to maximum concentration (t(max)) was delayed by 1.25 hours; maximum concentration (C(max)) and area under the curve (AUC) were increased, with reduced interindividual variability at higher doses of BAY 59-7939. Compared with baseline, BAY 59-7939 resulted in a relative increase in maximum prothrombin time (PT) prolongation of 44% (10 mg) and 53% (20 mg) in the fasted state, compared with 53% and 83% after food. Time to maximum PT prolongation was delayed by 0.5 to 1.5 hours after food, with no relevant influence of food type. No significant difference in C(max) and AUC was observed with coadministration of BAY 59-7939 and ranitidine or antacid.

  11. Molecular phylogeny, homology modeling, and molecular dynamics simulation of race-specific bacterial blight disease resistance protein (xa5) of rice: a comparative agriproteomics approach.

    PubMed

    Dehury, Budheswar; Sahu, Mousumi; Sarma, Kishore; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Sharma, Gauri Dutta; Choudhury, Manabendra Dutta; Barooah, Madhumita

    2013-08-01

    Rice (Oryza sativa L.), a model plant belonging to the family Poaceae, is a staple food for a majority of the people worldwide. Grown in the tropical and subtropical regions of the world, this important cereal crop is under constant and serious threat from both biotic and abiotic stresses. Among the biotic threats, Xanthomonas oryzae pv. oryzae, causing the damaging bacterial blight disease in rice, is a prominent pathogen. The xa5 gene in the host plant rice confers race-specific resistance to this pathogen. This recessive gene belongs to the Xa gene family of rice and encodes a gamma subunit of transcription factor IIA (TFIIAγ). In view of the importance of this gene in conferring resistance to the devastating disease, we reconstructed the phylogenetic relationship of this gene, developed a three-dimensional protein model, followed by long-term molecular dynamics simulation studies to gain a better understanding of the evolution, structure, and function of xa5. The modeled structure was found to fit well with the small subunit of TFIIA from human, suggesting that it may also act as a small subunit of TFIIA in rice. The model had a stable conformation in response to the atomic flexibility and interaction, when subjected to MD simulation at 20 nano second in aqueous solution. Further structural analysis of xa5 indicated that the protein retained its basic transcription factor function, suggesting that it might govern a novel pathway responsible for bacterial blight resistance. Future molecular docking studies of xa5 underway with its corresponding avirulence gene is expected to shed more direct light into plant-pathogen interactions at the molecular level and thus pave the way for richer agriproteomic insights.

  12. Factor 8 (F8) gene mutation profile of Turkish hemophilia A patients with inhibitors.

    PubMed

    Fidanci, Inanç D; Kavakli, Kaan; Uçar, Canan; Timur, Cetin; Meral, Adalet; Kilinç, Yurdanur; Sayilan, Hülya; Kazanci, Elif; Cağlayan, S Hande

    2008-07-01

    Factor VIII (FVIII) replacement therapy is ineffective in hemophilia A patients who develop alloantibodies (inhibitors) against FVIII. The type of factor 8 (F8) gene mutation, genes in the major histocompatibility complex loci, and also polymorphisms in IL-10 and tumor necrosis factor-alpha are the major predisposing factors for inhibitor formation. The present study was initiated to reveal the F8 gene mutation profile of 30 severely affected high-responder patients with inhibitor levels of more than 5 Bethesda U (BU)/ml and four low-responder patients with inhibitors less than 5 BU/ml. Southern blot and PCR analysis were performed to detect intron 22 and intron 1 inversions, respectively. Point mutations were screened by DNA sequence analysis of all coding regions, intron/exon boundaries, promoter and 3' UTR regions of the F8 gene. The prevalent mutation was the intron 22 inversion among the high-responder patients followed by large deletions, small deletions, and nonsense mutations. Only one missense and one splicing error mutation was seen. Among the low-responder patients, three single nucleotide deletions and one intron 22 inversion were found. All mutation types detected were in agreement with the severe hemophilia A phenotype, most likely leading to a deficiency of and predisposition to the development of alloantibodies against FVIII. It is seen that Turkish hemophilia A patients with major molecular defects have a higher possibility of developing inhibitors.

  13. Relation of factor VIII and IX inhibitors with ABO blood groups in 150 patients with haemophilia A and B.

    PubMed

    Torghabeh, Hassan Mansouri; Pourfathollah, Aliakbar; Shooshtari, Mahmood Mahmoodian; Yazdi, Zahra Rezaie

    2006-03-01

    Many investigations have proved relations between ABO blood groups with some diseases and factor VIII and von willebrand level in plasma. In this study we investigated a relation between ABO blood groups and factor VIII and IX inhibitors in 102 patients with haemophilia A and 48 patients with haemophilia B. The assay of inhibitor was done by Bethesda method. There were no relation between ABO blood groups and factor VIII and IX inhibitors.

  14. Antidotes to anthrax lethal factor intoxication. Part 1: Discovery of potent lethal factor inhibitors with in vivo efficacy.

    PubMed

    Jiao, Guan-Sheng; Kim, Seongjin; Moayeri, Mahtab; Cregar-Hernandez, Lynne; McKasson, Linda; Margosiak, Stephen A; Leppla, Stephen H; Johnson, Alan T

    2010-11-15

    Sub-nanomolar small molecule inhibitors of anthrax lethal factor have been identified using SAR and Merck L915 (4) as a model compound. One of these compounds (16) provided 100% protection in a rat lethal toxin model of anthrax disease.

  15. Arginine mimetic structures in biologically active antagonists and inhibitors.

    PubMed

    Masic, Lucija Peterlin

    2006-01-01

    Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides. L-Arginine is a guanidino group-containing basic amino acid, which is positively charged at neutral pH and is involved in many important physiological and pathophysiological processes. Many enzymes display a preference for the arginine residue that is found in many natural substrates and in synthetic inhibitors of many trypsin-like serine proteases, e.g. thrombin, factor Xa, factor VIIa, trypsin, and in integrin receptor antagonists, used to treat many blood-coagulation disorders. Nitric oxide (NO), which is produced by oxidation of L-arginine in an NADPH- and O(2)-dependent process catalyzed by isoforms of nitric oxide synthase (NOS), exhibits diverse roles in both normal and pathological physiologies and has been postulated to be a contributor to the etiology of various diseases. Development of NOS inhibitors as well as analogs and mimetics of the natural substrate L-arginine, is desirable for potential therapeutic use and for a better understanding of their conformation when bound in the arginine binding site. The guanidino residue of arginine in many substrates, inhibitors, and antagonists forms strong ionic interactions with the carboxylate of an aspartic acid moiety, which provides specificity for the basic amino acid residue in the active side. However, a highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is often associated with low selectivity and poor bioavailability after peroral application. Thus, significant effort is focused on the design and preparation of arginine mimetics that can confer selective inhibition for specific trypsin-like serine proteases and NOS inhibitors as well as integrin receptor antagonists and possess reduced basicity for enhanced oral bioavailability. This review will describe the survey of arginine mimetics designed to mimic the function of the

  16. Effect of tumor necrosis factorinhibitors on ambulatory 24-h blood pressure.

    PubMed

    Grossman, Chagai; Bornstein, Gil; Leibowitz, Avshalom; Ben-Zvi, Ilan; Grossman, Ehud

    2017-02-01

    Tumor necrosis factor alpha (TNF-α) inhibitors are increasingly being used in inflammatory rheumatic diseases (IRD). The risk of cardiovascular disease is elevated in patients with IRD and TNF-α inhibitors reduce this risk. We assessed whether the beneficial effect of TNF-α inhibitors on cardiovascular risk is mediated by blood pressure reduction. We measured blood pressure levels with 24-h ambulatory blood pressure measurements device in patients with IRD before and 3 months after treatment with TNF-α inhibitors. The study population consisted of 15 subjects (6 men; mean age 45.9 ± 14.1 years). Most patients had either rheumatoid arthritis or psoriatic arthritis and adalimumab was the most common TNF-α inhibitor used. Mean 24-h systolic and diastolic blood pressure levels remained the same after treatment (121 ± 12/66 ± 7 before and 123 ± 11/67 ± 10 mm Hg after; p = 0.88 and 0.66, respectively). The study demonstrates that TNF-α inhibitors have no effect on blood pressure levels.

  17. Inhibitors of propagation of coagulation: factors V and X

    PubMed Central

    Toschi, Vincenzo; Lettino, Maddalena

    2011-01-01

    Cardiovascular diseases are still the most important cause of morbidity and mortality in western countries and antithrombotic treatment is nowadays widely used. Drugs able to reduce coagulation activation are the treatment of choice for a number of arterial and/or venous thromboembolic conditions. Some of the drugs currently used for this purpose, such as heparins (UFH or LMWH) and VKA, have limitations consisting of a narrow therapeutic window and an unpredictable response with the need of laboratory monitoring in order to assess their efficacy and safety. These drawbacks have stimulated an active research aimed to develop new drugs able to act on single factors involved in the coagulation network, with predictable response. Intense experimental and clinical work on new drugs has focused on synthetic agents, which could preferably be administered orally and at fixed doses. The most advanced clinical development with new anticoagulants has been achieved for those inhibiting FXa and some of them, like fondaparinux, are already currently used in clinical practice. Other agents, such as rivaroxaban, apixaban, otamixaban and edoxaban are under development and have already been studied or are currently under investigation in large scale phase III clinical trials for prevention and treatment of venous thromboembolism, atrial fibrillation and acute coronary syndromes. Some of them have proved to be more effective than conventional therapy. Data on some agents inhibiting FVa are still preliminary and some of these drugs have so far been considered only in patients with disseminated intravascular coagulation secondary to sepsis. PMID:21545479

  18. IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII

    PubMed Central

    Matino, Davide; Gargaro, Marco; Santagostino, Elena; Di Minno, Matteo N.D.; Castaman, Giancarlo; Morfini, Massimo; Rocino, Angiola; Mancuso, Maria E.; Di Minno, Giovanni; Coppola, Antonio; Talesa, Vincenzo N.; Volpi, Claudia; Vacca, Carmine; Orabona, Ciriana; Iannitti, Rossana; Mazzucconi, Maria G.; Santoro, Cristina; Tosti, Antonella; Chiappalupi, Sara; Sorci, Guglielmo; Tagariello, Giuseppe; Belvini, Donata; Radossi, Paolo; Landolfi, Raffaele; Fuchs, Dietmar; Boon, Louis; Pirro, Matteo; Marchesini, Emanuela; Grohmann, Ursula; Puccetti, Paolo; Iorio, Alfonso; Fallarino, Francesca

    2015-01-01

    The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein. PMID:26426076

  19. Secretion of soluble complement inhibitors factor H and factor H-like protein (FHL-1) by ovarian tumour cells.

    PubMed

    Junnikkala, S; Hakulinen, J; Jarva, H; Manuelian, T; Bjørge, L; Bützow, R; Zipfel, P F; Meri, S

    2002-11-04

    We observed that the soluble complement regulators factor H and factor H-like protein were abundantly present in ascites samples as well as in primary tumours of patients with ovarian cancer. RT-PCR and immunoblotting analyses showed that the two complement inhibitors were constitutively produced by the ovarian tumour cell lines SK-OV-3 and Caov-3, but not PA-1 or SW626 cells. The amounts of factor H-like protein secreted were equal to those of factor H. This is exceptional, because e.g. in normal human serum the concentration of factor H-like protein is below 1/10th of that of factor H. In ascites samples the mean level of factor H-like protein (130+/-55 microg ml(-1)) was 5.5-fold higher than in normal human serum (24+/-3 microg ml(-1)). Ovarian tumour cells thus preferentially synthesise factor H-like protein, the alternatively spliced short variant of factor H. The tumour cells were found to bind both (125)I-labelled factor H and recombinant factor H-like protein to their surfaces. Surprisingly, the culture supernatants of all of the ovarian tumour cell lines studied, including those of PA-1 and SW626 that did not produce factor H/factor H-like protein, promoted factor I-mediated cleavage of C3b to inactive iC3b. Subsequently, the PA-1 and SW626 cell lines were found to secrete a soluble form of the membrane cofactor protein (CD46). Thus, our studies reveal two novel complement resistance mechanisms of ovarian tumour cells: (i) production of factor H-like protein and factor H and (ii) secretion of soluble membrane cofactor protein. Secretion of soluble complement inhibitors could protect ovarian tumour cells against humoral immune attack and pose an obstacle for therapy with monoclonal antibodies.

  20. Pharmacophore selection and redesign of non-nucleotide inhibitors of anthrax edema factor.

    PubMed

    Schein, Catherine H; Chen, Deliang; Ma, Lili; Kanalas, John J; Gao, Jian; Jimenez, Maria Estrella; Sower, Laurie E; Walter, Mary A; Gilbertson, Scott R; Peterson, Johnny W

    2012-11-08

    Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF), an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin's basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC) in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.

  1. Fast rotation of a subkilometer-sized near-Earth object 2011 XA{sub 3}

    SciTech Connect

    Urakawa, Seitaro; Ohtsuka, Katsuhito; Abe, Shinsuke; Ito, Takashi; Nakamura, Tomoki

    2014-05-01

    We present light curve observations and their multiband photometry for near-Earth object (NEO) 2011 XA{sub 3}. The light curve has shown a periodicity of 0.0304 ± 0.0003 days (= 43.8 ± 0.4 minutes). The fast rotation shows that 2011 XA{sub 3} is in a state of tension (i.e., a monolithic asteroid) and cannot be held together by self-gravitation. Moreover, the multiband photometric analysis indicates that the taxonomic class of 2011 XA{sub 3} is S-complex, or V-type. Its estimated effective diameter is 225 ± 97 m (S-complex) and 166 ± 63 m (V-type), respectively. Therefore, 2011 XA{sub 3} is a candidate for the second-largest, fast-rotating, monolithic asteroid. Moreover, the orbital parameters of 2011 XA{sub 3} are apparently similar to those of NEO (3200) Phaethon, but F/B-type. We computed the orbital evolutions of 2011 XA{sub 3} and Phaethon. However, the results of the computation and distinct taxonomy indicate that neither of the asteroids is of common origin.

  2. Yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors.

    PubMed

    Kim, Joungmok; Park, Hae-Chul; Gedi, Vinayakumar; Park, Hye-Yeon; Roberts, Arthur G; Atkins, William M; Yoon, Moon-Young

    2011-01-07

    Inhibitors of anthrax lethal factor (LF) are currently being sought as effective therapeutics for the treatment of anthrax. Here we report a novel screening approach for inhibitors of LF, a yeast-hybrid-based assay system in which the expression of reporter genes from a Gal4 promoter is repressed by LF proteolytic activity. Yeast cells were co-transformed with LF and a chimeric transcription factor that contains an LF substrate sequence inserted between the DNA-binding and activation domains of Gal4. In the resulting yeast cells, LF cleaves the substrate, thus inactivating the chimeric Gal4 and resulting in lack of expression of reporter genes. Compounds that inhibit LF cleavage of its substrate are identified by changes in reporter gene activity. Relative to in vitro screens for inhibitors of LF proteolytic activity, this screen has the advantage of excluding compounds that are toxic or non-permeable to eukaryotic cells. Additionally, the screen has the advantage of being fast, easy and cheap because exogenous LF and substrate are not needed. An initial chemical library screen with this system has identified four candidate inhibitors which were confirmed to inhibit LF protease activity in an in vitro assay. Furthermore, FBS-00831, one of the compounds identified, protects Raw 264.7 macrophages from anthrax lethal toxin and the possible binding site on LF was also evaluated by molecular docking.

  3. Vascular endothelial growth factor inhibitors: investigational therapies for the treatment of psoriasis

    PubMed Central

    Weidemann, Anja K; Crawshaw, Ania A; Byrne, Emily; Young, Helen S

    2013-01-01

    Psoriasis is a common inflammatory autoimmune condition in which environmental factors and genetic predisposition contribute to the development of disease in susceptible individuals. Angiogenesis is known to be a key pathogenic feature of psoriasis. Local and systemic elevation of vascular endothelial growth factor (VEGF)-A has been demonstrated in the skin and plasma of patients with psoriasis and is known to correlate with improvement following some traditional psoriasis treatments. A number of VEGF inhibitors are licensed for the treatment of malignancies and eye disease and isolated case reports suggest that some individuals with psoriasis may improve when exposed to these agents. The small number of cases and lack of unified reporting measures makes it difficult to draw generalizations and underline the heterogeneity of psoriasis as a disease entity. Though not yet licensed for the treatment of psoriasis in humans, experimental data supports the potential of VEGF inhibitors to influence relevant aspects of human cell biology (such as endothelial cell differentiation) and to improve animal models of skin disease. Given the multi-factorial nature of psoriasis it is unlikely that VEGF inhibitors will be effective in all patients, however they have the potential to be a valuable addition to the therapeutic arsenal in selected cases. Current VEGF inhibitors in clinical use are associated with a number of potentially serious side effects including hypertension, left ventricular dysfunction, and gastrointestinal perforation. Such risks require careful consideration in psoriasis populations particularly in light of growing concerns linking psoriasis to increased cardiovascular risk. PMID:24101875

  4. Prolonged Activated Partial Thromboplastin Time: Difficulties in Discriminating Coexistent Factor VIII Inhibitor and Lupus Anticoagulant.

    PubMed

    Ames, Paul R J; Graf, Maria; Archer, Jeremy; Scarpato, Nicola; Iannaccone, Luigi

    2015-03-01

    To review the diagnostic difficulties of a prolonged activated partial thromboplastin time (aPTT) when 2 inhibitors with opposite clinical presentations coexist, we searched MEDLINE from January 1970 to November 2013 using acquired, factor VIII (FVIII), factor IX, hemophilia A and B, inhibitor, lupus anticoagulant (LA), antiphospholipid, anticardiolipin, anti-β2-glycoprotein I, antibodies, syndrome, bleeding, and thrombosis. We identified 13 articles for a total of 15 cases of possible coexistence of FVIII inhibitor and LA. The presenting clinical manifestation was thrombosis in 6 cases and bleeding in 9 cases. Activated partial thromboplastin time was the presenting laboratory abnormality in all cases, and first-line investigations suggested the coexistence of LA and acquired FVIII inhibitor. None of the articles addressed the diagnostic accuracy of the screening tests by performing "second line" assays. We reviewed the diagnostic pitfalls of the cases under study and provide some guidance for alternative tests when facing a prolonged aPTT that may have a double meaning.

  5. ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy.

    PubMed

    Erpenbeck, Luise; Demers, Melanie; Zsengellér, Zsuzsanna K; Gallant, Maureen; Cifuni, Stephen M; Stillman, Isaac E; Karumanchi, S Ananth; Wagner, Denisa D

    2016-01-01

    Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor-related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor-related TMA. ADAMTS13(-/-) mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor-related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13(-/-) mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.

  6. Inhibition of the activation of Hageman factor (factor XII) by peripheral blood cells.

    PubMed Central

    Ratnoff, O D; Emanuelson, M M; Ziats, N P

    1987-01-01

    Suspensions of peripheral blood mononuclear cells (PBMC), monocytes, T or B lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a property not shared by erythrocytes. PBMC also inhibited HF activation by glass or sulfatides. Contaminating platelets may have contributed to inhibition by PBMC. Elaboration of agents inhibiting HF activation required metabolically active cells. The inhibitor(s) in PBMC supernates were not identified with known agents, but had properties of a nonenzymatic protein. PBMC supernates did not contain fibrinogen, nor alter the thrombin, prothrombin, or partial thromboplastin times of normal plasma, amidolysis by activated plasma thromboplastin antecedent (Factor XIa) or activated Stuart factor (Factor Xa) or esterolysis by C1 (C1 esterase); they inhibited plasmin minimally. These experiments suggest that peripheral blood cells may impede intravascular coagulation. Whether this property helps maintain the fluidity of blood is unclear. PMID:3498741

  7. Acquired Factor V Inhibitors in a Patient with End-stage Renal Disease

    PubMed Central

    Kitazawa, Atsushi; Misawa, Hideo; Nagahori, Katsuhiro; Koda, Ryo; Yoshino, Atsunori; Kawamoto, Shinya; Takeda, Tetsuro

    2016-01-01

    We report a case of acquired factor V inhibitors (AFVIs) in a patient with end-stage renal disease receiving warfarin therapy for atrial fibrillation. A 72-year-old Japanese man was admitted to our hospital complaining of tarry stools and abdominal pain. The laboratory findings revealed eosinophilia (52.1%), prolonged activated partial thromboplastin time (APTT) (98 s), PT (84 s), a factor V (FV) activity of <3%, and an FV inhibitor level of 6 Bethesda units/mL. After administration of prednisolone was started, his coagulation findings improved. However, his renal failure progressed, and he ultimately required chronic hemodialysis. This is the first case of AFVIs in a patient starting hemodialysis for end-stage renal disease. PMID:27904118

  8. Acquired Factor V Inhibitors in a Patient with End-stage Renal Disease.

    PubMed

    Kitazawa, Atsushi; Misawa, Hideo; Nagahori, Katsuhiro; Koda, Ryo; Yoshino, Atsunori; Kawamoto, Shinya; Takeda, Tetsuro

    We report a case of acquired factor V inhibitors (AFVIs) in a patient with end-stage renal disease receiving warfarin therapy for atrial fibrillation. A 72-year-old Japanese man was admitted to our hospital complaining of tarry stools and abdominal pain. The laboratory findings revealed eosinophilia (52.1%), prolonged activated partial thromboplastin time (APTT) (98 s), PT (84 s), a factor V (FV) activity of <3%, and an FV inhibitor level of 6 Bethesda units/mL. After administration of prednisolone was started, his coagulation findings improved. However, his renal failure progressed, and he ultimately required chronic hemodialysis. This is the first case of AFVIs in a patient starting hemodialysis for end-stage renal disease.

  9. Identification of inhibitors for vascular endothelial growth factor receptor by using dynamic combinatorial chemistry.

    PubMed

    Yang, Zhao; Fang, Zheng; He, Wei; Wang, Zhixiang; Gan, Haifeng; Tian, Qitao; Guo, Kai

    2016-04-01

    The novel analysis method consisting of size-exclusion chromatography (SEC) and HRMS analysis was firstly applied in the discovery of potential inhibitors towards cancer drug targets. With vascular endothelial growth factor receptor (VEGFR-2) as a target, dynamic combinatorial libraries (DCLs) were prepared by reacting aldehydes with amines. Four sensitive binders targeted VEGFR-2 were directly isolated from the library. Antitumor activity test in vitro and inhibition experiments toward angiogenesis were also carried out.

  10. Improved coagulation and hemostasis in hemophilia with inhibitors by combinations of superFactor Va and Factor VIIa

    PubMed Central

    Bhat, Vikas; von Drygalski, Annette; Gale, Andrew J.; Griffin, John H.; Mosnier, Laurent O.

    2015-01-01

    Bypassing inhibitors in hemophilia patients is limited to activated (a) Factor(F)VII products. We introduced “FVa activity augmentation” as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilizing properties of superFVa and recombinant human (rh)FVIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in hemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. superFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e.g., 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalize clot lysis times could be reduced by 100-fold (e.g., from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In hemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of hemophilia with inhibitors and it merits further evaluation for new bypassing strategies. PMID:26466980

  11. Small-molecule inhibitors of lethal factor protease activity protect against anthrax infection.

    PubMed

    Moayeri, Mahtab; Crown, Devorah; Jiao, Guan-Sheng; Kim, Seongjin; Johnson, Alan; Leysath, Clinton; Leppla, Stephen H

    2013-09-01

    Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

  12. A molecular modeling study of inhibitors of nuclear factor kappa-B (p50) DNA binding

    NASA Astrophysics Data System (ADS)

    Pande, Vineet; Sharma, Rakesh K.; Inoue, Jun-Ichiro; Otsuka, Masami; Ramos, Maria J.

    2003-12-01

    Nuclear Factor-kappa B (NF-κB) is an inducible transcription factor of the Rel family, and is sequestered in the cytoplasm by the IκB family of proteins. NF-κB can exist in several dimeric forms, but the p50/p65 heterodimer is the predominant one. Activation of NF-κB by a range of stimuli including viral products, and oxidative stress, leads to phosphorylation and proteasome dependent degradation of IκB, leading to the release of free NF-κB. This free NF-κB then binds to its target sites (κB sites in the DNA) to initiate transcription. These κB sites are also present in the Long Terminal Repeat (LTR) of HIV-1, and hence NF-κB (p50 subunit) binding to LTR-DNA is critical in viral replication. Targeting direct p50-DNA binding, in this regard, is a novel approach to design anti-HIV gene expression inhibitors, which do not have the problem of resistance unlike in other anti-HIV strategies. The present study is a part of our search for leads for the specific inhibition of p50-DNA binding. We have been experimentally studying different types of these inhibitors, and in this work, we attempted to get a common definition of their structural mechanism onto p50-DNA binding. Using three different classes of inhibitors, we modelled their association with the DNA-Binding Region (DBR) of the p50 subunit of NF-κB. Docking studies were carried out using a genetic algorithm based program (GOLD). Further, to compare electrostatic complementarity in the association of the inhibitors with the DBR, Molecular Electrostatic Potentials (MEPs) were generated for the DBR and each inhibitor. The results of docking revealed a strong network of hydrogen bonding interactions for every active inhibitor, and the contrary for the less active ones. Further, the MEPs revealed that the DBR of p50 represents a surface of electropositive potential, and the active inhibitors represent a complementary electronegative surface. With the present modelling study we conclude that the principal

  13. A molecular modeling study of inhibitors of nuclear factor kappa-B (p50)--DNA binding.

    PubMed

    Pande, Vineet; Sharma, Rakesh K; Inoue, Jun-Ichiro; Otsuka, Masami; Ramos, Maria J

    2003-12-01

    Nuclear Factor-kappa B (NF-kappaB) is an inducible transcription factor of the Rel family, and is sequestered in the cytoplasm by the IkappaB family of proteins. NF-kappaB can exist in several dimeric forms, but the p50/p65 heterodimer is the predominant one. Activation of NF-kappaB by a range of stimuli including viral products, and oxidative stress, leads to phosphorylation and proteasome dependent degradation of IkappaB, leading to the release of free NF-kappaB. This free NF-kappaB then binds to its target sites (KB sites in the DNA) to initiate transcription. These kappaB sites are also present in the Long Terminal Repeat (LTR) of HIV-1, and hence NF-kappaB (p50 subunit) binding to LTR-DNA is critical in viral replication. Targeting direct p50-DNA binding, in this regard, is a novel approach to design anti-HIV gene expression inhibitors, which do not have the problem of resistance unlike in other anti-HIV strategies. The present study is a part of our search for leads for the specific inhibition of p50-DNA binding. We have been experimentally studying different types of these inhibitors, and in this work, we attempted to get a common definition of their structural mechanism onto p50-DNA binding. Using three different classes of inhibitors, we modelled their association with the DNA-Binding Region (DBR) of the p50 subunit of NF-kappaB. Docking studies were carried out using a genetic algorithm based program (GOLD). Further, to compare electrostatic complementarity in the association of the inhibitors with the DBR, Molecular Electrostatic Potentials (MEPs) were generated for the DBR and each inhibitor. The results of docking revealed a strong network of hydrogen bonding interactions for every active inhibitor, and the contrary for the less active ones. Further, the MEPs revealed that the DBR of p50 represents a surface of electropositive potential, and the active inhibitors represent a complementary electronegative surface. With the present modelling study we

  14. Saururus cernuus Lignans - Potent Small Molecule Inhibitors of Hypoxia-Inducible Factor-1

    PubMed Central

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R.; Zhang, Lei; Bruick, Richard K.; Mohammed, Kaleem A.; Agarwal, Ameeta K.; Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors. PMID:15967416

  15. Saururus cernuus lignans--potent small molecule inhibitors of hypoxia-inducible factor-1.

    PubMed

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R; Zhang, Lei; Bruick, Richard K; Mohammed, Kaleem A; Agarwal, Ameeta K; Nagle, Dale G; Zhou, Yu-Dong

    2005-08-05

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.

  16. Effect of nadroparin on anti-Xa activity during nocturnal hemodialysis

    PubMed Central

    Buitenwerf, Edward; Risselada, Arne J.; van Roon, Eric N.; Veeger, Nic J.G.M.; Hemmelder, Marc H.

    2015-01-01

    Background Nadroparin is used during hemodialysis to prevent clotting of the extra corporeal system. During nocturnal hemodialysis patients receive an increased dosage of nadroparin compared to conventional hemodialysis. We tested whether the prescribed dosage regimen of nadroparin, according to Dutch guidelines, causes accumulation of nadroparin. Methods Anti-Xa levels were used as an indicator of nadroparin accumulation. Anti-Xa was measured photometrically in 13 patients undergoing nocturnal hemodialysis for 4 nights a week. Nadroparin was administered according to Dutch dosage guidelines. We assessed anti-Xa levels at 4 time points during 1 dialysis week: before the start of the first dialysis session of the week (baseline), prior to (T1) and after the last dialysis session of the week (T2) and before the first dialysis of the following week (T3). Results Patients received 71–95 IU/kg at the start of dialysis and another 50% of the initial dosage after 4 h with a total cumulative dosage of 128 ± 24 IU/kg. Anti-Xa levels increased from 0.017 at baseline to 0.019 at T1 (p = 0.03). Anti-Xa levels were 0.419 ± 0.252 IU/ml at T2 (p < 0.001 vs baseline and T1), whereas anti-Xa levels were not changed at T3 compared to baseline. Conclusion Dosing of nadroparin according to Dutch guidelines in patients on nocturnal hemodialysis does not lead to accumulation of nadroparin. We therefore consider the Dutch dosage guidelines for nadroparin an effective and safe strategy. General significance This article is the first to present data on anti-Xa activity during nocturnal hemodialysis which is a widely used and potentially dangerous therapy. PMID:26672512

  17. Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein.

    PubMed

    Mast, Alan E

    2016-01-01

    Tissue factor (TF) pathway inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response. Alternatively spliced isoforms of TFPI are differentially expressed by endothelial cells and human platelets and plasma. The TFPIβ isoform localizes to the endothelium surface where it is a potent inhibitor of TF-factor VIIa complexes that initiate blood coagulation. The TFPIα isoform is present in platelets. TFPIα contains a stretch of 9 amino acids nearly identical to those found in the B-domain of factor V that are well conserved in mammals. These amino acids provide exosite binding to activated factor V, which allows for TFPIα to inhibit prothrombinase during the initiation phase of blood coagulation. Endogenous inhibition at this point in the coagulation cascade was only recently recognized and has provided a biochemical rationale to explain the pathophysiological mechanisms underlying several clinical disorders. These include the east Texas bleeding disorder that is caused by production of an altered form of factor V with high affinity for TFPI and a paradoxical procoagulant effect of heparins. In addition, these findings have led to ideas for pharmacological targeting of TFPI that may reduce bleeding in hemophilia patients.

  18. Highly predictive support vector machine (SVM) models for anthrax toxin lethal factor (LF) inhibitors.

    PubMed

    Zhang, Xia; Amin, Elizabeth Ambrose

    2016-01-01

    Anthrax is a highly lethal, acute infectious disease caused by the rod-shaped, Gram-positive bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF), a zinc metalloprotease secreted by the bacilli, plays a key role in anthrax pathogenesis and is chiefly responsible for anthrax-related toxemia and host death, partly via inactivation of mitogen-activated protein kinase kinase (MAPKK) enzymes and consequent disruption of key cellular signaling pathways. Antibiotics such as fluoroquinolones are capable of clearing the bacilli but have no effect on LF-mediated toxemia; LF itself therefore remains the preferred target for toxin inactivation. However, currently no LF inhibitor is available on the market as a therapeutic, partly due to the insufficiency of existing LF inhibitor scaffolds in terms of efficacy, selectivity, and toxicity. In the current work, we present novel support vector machine (SVM) models with high prediction accuracy that are designed to rapidly identify potential novel, structurally diverse LF inhibitor chemical matter from compound libraries. These SVM models were trained and validated using 508 compounds with published LF biological activity data and 847 inactive compounds deposited in the Pub Chem BioAssay database. One model, M1, demonstrated particularly favorable selectivity toward highly active compounds by correctly predicting 39 (95.12%) out of 41 nanomolar-level LF inhibitors, 46 (93.88%) out of 49 inactives, and 844 (99.65%) out of 847 Pub Chem inactives in external, unbiased test sets. These models are expected to facilitate the prediction of LF inhibitory activity for existing molecules, as well as identification of novel potential LF inhibitors from large datasets.

  19. Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

    PubMed

    El-Mowafi, S A; Sineva, E; Alumasa, J N; Nicoloff, H; Tomsho, J W; Ades, S E; Keiler, K C

    2015-01-01

    Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

  20. Identification and analysis of the tissue factor pathway inhibitor 2 of Sciaenops ocellatus.

    PubMed

    Zhang, Min; Xiao, Zhi-zhong; Sun, Li

    2011-01-01

    Tissue factor pathway inhibitor 2 (TFPI-2) is a structural homologue of TFPI, a potent inhibitor of tissue factor (TF)-mediated coagulation. Although TFPI-2 has been identified at sequence level in several fish species, no study on piscine TFPI-2 has been documented. In this report, we identified and analyzed a TFPI-2 homologue, SoTFPI2, from red drum Sciaenops ocellatus. The open reading frame of SoTFPI2 is 681 bp, which encodes a 226-residue protein that shares 59.2-82.3% overall sequence identities with known fish TFPI-2. SoTFPI2 possesses three tandem Kunitz domains and is negatively charged at the N-terminus and positively charged at the C-terminus. Expression of SoTFPI2 was detected, in increasing order, in spleen, muscle, gill, brain, liver, kidney, blood, and heart. Bacterial challenge and lipopolysaccharide treatment significantly upregulated SoTFPI2 expression in kidney in time-dependent manners. Recombinant SoTFPI2 purified from Escherichia coli inhibits the proteolytic activity of trypsin and exhibits bactericidal effect on a fish pathogen. Take together, these results indicate that SoTFPI2 is a biologically active serine protease inhibitor with antibacterial property and is likely to play a role in anti-bacterial infection.

  1. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

    PubMed

    Kitamura, Seiya; Hvorecny, Kelli L; Niu, Jun; Hammock, Bruce D; Madden, Dean R; Morisseau, Christophe

    2016-05-26

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

  2. A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration

    PubMed Central

    Jang, Kusik; Kim, Inhyub; Kim, Bo-Kyung; Lee, Kyeong; Won, Misun

    2016-01-01

    We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells. PMID:27611801

  3. Hypoxia inducible factor 1α expression and effects of its inhibitors in canine lymphoma

    PubMed Central

    KAMBAYASHI, Satoshi; IGASE, Masaya; KOBAYASHI, Kosuke; KIMURA, Ayana; SHIMOKAWA MIYAMA, Takako; BABA, Kenji; NOGUCHI, Shunsuke; MIZUNO, Takuya; OKUDA, Masaru

    2015-01-01

    Hypoxic conditions in various cancers are believed to relate with their malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major regulator of the response to low oxygen. In this study, we examined HIF-1α expression in canine lymphoma using cell lines and clinical samples and found that these cells expressed HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol, suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor. Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine lymphoma. PMID:26050843

  4. 3D/3D registration of coronary CTA and biplane XA reconstructions for improved image guidance

    SciTech Connect

    Dibildox, Gerardo Baka, Nora; Walsum, Theo van; Punt, Mark; Aben, Jean-Paul; Schultz, Carl; Niessen, Wiro

    2014-09-15

    Purpose: The authors aim to improve image guidance during percutaneous coronary interventions of chronic total occlusions (CTO) by providing information obtained from computed tomography angiography (CTA) to the cardiac interventionist. To this end, the authors investigate a method to register a 3D CTA model to biplane reconstructions. Methods: The authors developed a method for registering preoperative coronary CTA with intraoperative biplane x-ray angiography (XA) images via 3D models of the coronary arteries. The models are extracted from the CTA and biplane XA images, and are temporally aligned based on CTA reconstruction phase and XA ECG signals. Rigid spatial alignment is achieved with a robust probabilistic point set registration approach using Gaussian mixture models (GMMs). This approach is extended by including orientation in the Gaussian mixtures and by weighting bifurcation points. The method is evaluated on retrospectively acquired coronary CTA datasets of 23 CTO patients for which biplane XA images are available. Results: The Gaussian mixture model approach achieved a median registration accuracy of 1.7 mm. The extended GMM approach including orientation was not significantly different (P > 0.1) but did improve robustness with regards to the initialization of the 3D models. Conclusions: The authors demonstrated that the GMM approach can effectively be applied to register CTA to biplane XA images for the purpose of improving image guidance in percutaneous coronary interventions.

  5. Tumor necrosis factor-alpha inhibitors suppress CCL2 chemokine in monocytes via epigenetic modification.

    PubMed

    Lin, Yi-Ching; Lin, Yu-Chih; Huang, Ming-Yii; Kuo, Po-Lin; Wu, Cheng-Chin; Lee, Min-Sheng; Hsieh, Chong-Chao; Kuo, Hsuan-Fu; Kuo, Chang-Hung; Tsai, Wen-Chan; Hung, Chih-Hsing

    2017-03-01

    The treatment of rheumatoid arthritis (RA) with tumor necrosis factor-alpha (TNF-α) inhibitors could lead to adverse effects. Therefore, the identification of downstream therapeutic targets is important. Monocyte chemoattractant protein-1 (MCP-1, also called CCL2) is related to RA disease activity, and epigenetic modifications are hypothesized to regulate gene expression in RA pathogenesis. We studied the effects of two TNF-α inhibitors, etanercept and adalimumab, on CCL2 expression and the potentially associated intracellular mechanisms, including epigenetic regulation. Etanercept and adalimumab decreased CCL2 production in THP-1 cells and human primary monocytes, as detected using enzyme-linked immunosorbent assays, and these changes in the CCL2 levels were independent of the TNF-α levels. Etanercept and adalimumab suppressed mitogen-activated protein kinase (MAPK) phospho-p38, phospho-JNK, phospho-ERK and nuclear factor-κB (NF-κB) phospho-p65, as demonstrated using western blot analyses. The investigation of epigenetic modifications using chromatin immunoprecipitation revealed that etanercept and adalimumab down-regulated acetylation of histone (H)3 and H4 in the CCL2 promoter region by decreasing the recruitment of the NF-κB associated acetyltransferases p300, CBP and PCAF. Etanercept and adalimumab also down-regulated trimethylation of H3K4, H3K27, H3K36 and H3K79 in the CCL2 promoter region by decreasing the expression of the related methyltransferases WDR5 and Smyd2. We demonstrated that TNF-α inhibitors exert immunomodulatory effects on CCL2 expression in human monocytes via MAPKs, NF-κB and epigenetic modifications. These findings broaden the mechanistic knowledge related to TNF-α inhibitors and provide novel therapeutic targets for RA.

  6. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

    PubMed Central

    Tucker, Erik I.; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D.; Gailani, Dave; Gruber, András; McCarty, Owen J. T.

    2015-01-01

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  7. Tissue factor and tissue factor pathway inhibitor as key regulators of global hemostasis: measurement of their levels in coagulation assays.

    PubMed

    Kasthuri, Raj S; Glover, Sam L; Boles, Jeremiah; Mackman, Nigel

    2010-10-01

    The tissue factor (TF)/factor (F)VIIa complex is the primary initiator of coagulation in vivo. Tissue factor pathway inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Deficiencies of either TF or TFPI have not been reported in humans, and a complete absence of either of these two proteins in mice is embryonically lethal. To maintain normal hemostasis, levels of TF and TFPI need to be balanced. Increased levels of TF can overwhelm the inhibitory capacity of TFPI, resulting in thrombosis. Decreased levels of TF are associated with bleeding. Global assays of coagulation are defined as tests capable of evaluating all components of the clotting cascade that are present in plasma. In these tests the thrombogenic surface is either provided by platelets or exogenous phospholipids. Clotting assays currently used in clinical practice are not designed to measure endogenous levels of TF and TFPI. Therefore, there is a need to develop sensitive and specific assays for measuring levels of functional TF and TFPI in whole blood and plasma. These assays could be useful in patient management in many scenarios.

  8. Molecular modeling studies of [6,6,5] Tricyclic Fused Oxazolidinones as FXa inhibitors using 3D-QSAR, Topomer CoMFA, molecular docking and molecular dynamics simulations.

    PubMed

    Xu, Cheng; Ren, Yujie

    2015-10-15

    Coagulation factor Xa (Factor Xa, FXa) is a particularly promising target for novel anticoagulant therapy. The first oral factor Xa inhibitor has been approved in the EU and Canada in 2008. In this work, 38 [6,6,5] Tricyclic Fused Oxazolidinones were studied using a combination of molecular modeling techniques including three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, molecular dynamics and Topomer CoMFA (comparative molecular field analysis) were used to build 3D-QSAR models. The results show that the best CoMFA model has q(2)=0.511 and r(2)=0.984, the best CoMSIA (comparative molecular similarity indices analysis) model has q(2)=0.700 and r(2)=0.993 and the Topomer CoMFA analysis has q(2)=0.377 and r(2)=0.886. The results indicated the steric, hydrophobic, H-acceptor and electrostatic fields play key roles in models. Molecular docking and molecular dynamics explored the binding relationship of the ligand and the receptor protein.

  9. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

    PubMed

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2015-08-01

    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  10. Antiplatelet agents affecting the interaction of Tissue Factor-Factor VIIa complex with Factor X in a continuous-flow reactor.

    PubMed

    Gir, S; Reavis, R; Turitto, V T; Gollamudi, R

    1996-02-01

    The purpose of the present study was to examine the role of antithrombotic agents in the activation of Factor X in the presence of the Tissue Factor-Factor VIIa (TF-VIIa) complex in a continuous-flow reactor. Tissue Factor immobilized in a phospholipid bilayer on the inner surface of a capillary tube (internal diameter = 0.27 mm) was exposed to a perfusate containing Factors VIIa and X flowing at a flow rate of 12.7 microliters/min, corresponding to a wall shear rate of 100 s-1. Factor Xa (the activated form of Factor X) in the effluent was determined by a chromogenic assay. The effectiveness of two platelet aggregation inhibitors, alpha,alpha'-bis-[3-(N,N-diethylcarbamoyl)piperidino-p-xylene dihydrobromide (A-1) and alpha,alpha'-bis-[3-N-benzyl-N-methylcarbamoyl)piperidino]-p-xylen e dihydrobromide (A-4) in inhibiting Factor X activation is reported here. The results suggest that the Tissue Factor pathway, mediated through TF-VIIa complex, produces significantly lower levels of Factor Xa in the presence of compounds A-1 and A-4. On the basis of these findings, it appears that the anticoagulation action of these compounds reinforces their platelet aggregation-inhibitory properties. These carbamoylpiperidines (nipecotamides) therefore appear to be useful antithrombotic agents.

  11. Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

    PubMed Central

    Ziegler, David S.; Wright, Renee D.; Kesari, Santosh; Lemieux, Madeleine E.; Tran, Mary A.; Jain, Monish; Zawel, Leigh; Kung, Andrew L.

    2008-01-01

    Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM. PMID:18677408

  12. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

    PubMed Central

    Wilson, Timothy R.; Fridlyand, Jane; Yan, Yibing; Penuel, Elicia; Burton, Luciana; Chan, Emily; Peng, Jing; Lin, Eva; Wang, Yulei; Sosman, Jeff; Ribas, Antoni; Li, Jiang; Moffat, John; Sutherlin, Daniel P.; Koeppen, Hartmut; Merchant, Mark; Neve, Richard; Settleman, Jeff

    2013-01-01

    Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy1. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance2,3. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase4. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK)5. Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma6 or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-‘addicted’ human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases. PMID:22763448

  13. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    PubMed Central

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-01-01

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13. PMID:25697265

  14. Dermatologic Reactions to Targeted Therapy: A Focus on Epidermal Growth Factor Receptor Inhibitors and Nursing Care.

    PubMed

    Barton-Burke, Margaret; Ciccolini, Kathryn; Mekas, Maria; Burke, Sean

    2017-03-01

    Cancer treatments usually have side effects of bone marrow depression, mucositis, hair loss, and gastrointestinal issues. Rarely do we think of skin side effects until patients have been treated successfully with epidermal growth factor receptor inhibitors (EGFRi). Those reactions include papulopustular rash, hair changes, radiation dermatitis enhancement, pruritus, mucositis, xerosis, fissures, and paronychia. This article discusses the common skin reactions seen when using EGFRi and presents an overview of skin as the largest and important organ of the body, including an overview of skin assessment, pathophysiology of the skin reactions, nursing care involved, and introduction to oncodermatology.

  15. Discovery of Novel Human Epidermal Growth Factor Receptor-2 Inhibitors by Structure-based Virtual Screening

    PubMed Central

    Shi, Zheng; Yu, Tian; Sun, Rong; Wang, Shan; Chen, Xiao-Qian; Cheng, Li-Jia; Liu, Rong

    2016-01-01

    Background: Human epidermal growth factor receptor-2 (HER2) is a trans-membrane receptor like protein, and aberrant signaling of HER2 is implicated in many human cancers, such as ovarian cancer, gastric cancer, and prostate cancer, most notably breast cancer. Moreover, it has been in the spotlight in the recent years as a promising new target for therapy of breast cancer. Objective: Since virtual screening has become an integral part of the drug discovery process, it is of great significant to identify novel HER2 inhibitors by structure-based virtual screening. Materials and Methods: In this study, we carried out a series of elegant bioinformatics approaches, such as virtual screening and molecular dynamics (MD) simulations to identify HER2 inhibitors from Food and Drug Administration-approved small molecule drug as potential “new use” drugs. Results: Molecular docking identified top 10 potential drugs which showed spectrum affinity to HER2. Moreover, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) might exert potential inhibitory effects against HER2-targeted anti-breast cancer therapeutics. Conclusion: Together, our findings may provide successful application of virtual screening studies in the lead discovery process, and suggest that our discovered small molecules could be effective HER2 inhibitor candidates for further study. SUMMARY A series of elegant bioinformatics approaches, including virtual screening and molecular dynamics (MD) simulations were took advantage to identify human epidermal growth factor receptor-2 (HER2) inhibitors. Molecular docking recognized top 10 candidate compounds, which showed spectrum affinity to HER2. Further, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) in candidate compounds were identified as potential “new use” drugs against HER2-targeted anti-breast cancer therapeutics. Abbreviations used: HER2: Human epidermal growth factor receptor-2

  16. Genetic engineering of the Xa10 promoter for broad-spectrum and durable resistance to Xanthomonas oryzae pv. oryzae.

    PubMed

    Zeng, Xuan; Tian, Dongsheng; Gu, Keyu; Zhou, Zhiyun; Yang, Xiaobei; Luo, Yanchang; White, Frank F; Yin, Zhongchao

    2015-09-01

    Many pathovars of plant pathogenic bacteria Xanthomonas species inject transcription activator-like (TAL) effectors into plant host cells to promote disease susceptibility or trigger disease resistance. The rice TAL effector-dependent disease resistance gene Xa10 confers narrow-spectrum race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease in rice. To generate broad-spectrum and durable resistance to Xoo, we developed a modified Xa10 gene, designated as Xa10(E5) . Xa10(E5) has an EBE-amended promoter containing 5 tandemly arranged EBEs each responding specifically to a corresponding virulent or avirulent TAL effector and a stable transgenic rice line containing Xa10(E5) was generated in the cultivar Nipponbare. The Xa10(E5) gene was specifically induced by Xoo strains that harbour the corresponding TAL effectors and conferred TAL effector-dependent resistance to the pathogens at all developmental stages of rice. Further disease evaluation demonstrated that the Xa10(E5) gene in either Nipponbare or 9311 genetic backgrounds provided broad-spectrum disease resistance to 27 of the 28 Xoo strains collected from 11 countries. The development of Xa10(E5) and transgenic rice lines provides new genetic materials for molecular breeding of rice for broad-spectrum and durable disease resistance to bacterial blight.

  17. Characterization and expression analysis of two cDNAs encoding Xa1 and oxysterol binding proteins in sorghum (Sorghum bicolor)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using suppression subtractive hybridization (SSH) and subsequent microarray analysis, expression profiles of sorghum genes responsive to greenbug phloem-feeding were obtained and identified. Among the profiles, two cDNAs designated to MM73 and MM95 were identified to encode Xa1 (Xa1) and oxysterol ...

  18. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates murine colitis

    PubMed Central

    Gupta, Ram; Chaudhary, Anita R; Shah, Binita N; Jadhav, Avinash V; Zambad, Shitalkumar P; Gupta, Ramesh Chandra; Deshpande, Shailesh; Chauthaiwale, Vijay; Dutt, Chaitanya

    2014-01-01

    Background and aim Mucosal healing in inflammatory bowel disease (IBD) can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF) has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn’s disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor ameliorates IBD in disease models. These findings highlight the potential of TRC160334 for its clinical application in the treatment of IBD. PMID:24493931

  19. Natural Product-Based Inhibitors of Hypoxia-Inducible Factor-1 (HIF-1)

    PubMed Central

    Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    The transcription factor hypoxia-inducible factor-1 (HIF-1) regulates the expression of more than 70 genes involved in cellular adaptation and survival under hypoxic stress. Activation of HIF-1 is associated with numerous physiological and pathological processes that include tumorigenesis, vascular remodeling, inflammation, and hypoxia/ischemia-related tissue damage. Clinical studies suggested that HIF-1 activation correlates directly with advanced disease stages and treatment resistance among cancer patients. Preclinical studies support the inhibition of HIF-1 as a major molecular target for antitumor drug discovery. Considerable effort is underway, in government laboratories, industry and academia, to identify therapeutically useful small molecule HIF-1 inhibitors. Natural products (low molecular weight organic compounds produced by plants, microbes, and animals) continue to play a major role in modern antitumor drug discovery. Most of the compounds discovered to inhibit HIF-1 are natural products or synthetic compounds with structures that are based on natural product leads. Natural products have also served a vital role as molecular probes to elucidate the pathways that regulate HIF-1 activity. Natural products and natural product-derived compounds that inhibit HIF-1 are summarized in light of their biological source, chemical class, ancd effect on HIF-1 and HIF-mediated gene regulation. When known, the mechanism(s) of action of HIF-1 inhibitors are described. Many of the substances found to inhibit HIF-1 are non-druggable compounds that are too cytotoxic to serve as drug leads. The application of high-throughput screening methods, complementary molecular-targeted assays, and structurally diverse chemical libraries hold promise for the discovery of therapeutically useful HIF-1 inhibitors. PMID:16515532

  20. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

    PubMed

    Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

    2001-01-01

    We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

  1. Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients.

    PubMed

    Pinto, P; Ghosh, K; Shetty, S

    2012-09-01

    Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1β, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients.

  2. Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas.

    PubMed

    Roskoski, Robert

    2017-03-19

    One Von Hippel-Lindau (VHL) tumor suppressor gene is lost in most renal cell carcinomas while the nondeleted allele exhibits hypermethylation-induced inactivation or inactivating somatic mutations. As a result of these genetic modifications, there is an increased production of VEGF-A and pro-angiogenic growth factors in this disorder. The important role of angiogenesis in the pathogenesis of renal cell carcinomas and other tumors has focused the attention of investigators on the biology of VEGFs and VEGFR1-3 and to the development of inhibitors of the intricate and multifaceted angiogenic pathways. VEGFR1-3 contain an extracellular segment with seven immunoglobulin-like domains, a transmembrane segment, a juxtamembrane segment, a protein kinase domain with an insert of about 70 amino acid residues, and a C-terminal tail. VEGF-A stimulates the activation of preformed VEGFR2 dimers by the auto-phosphorylation of activation segment tyrosines followed by the phosphorylation of additional protein-tyrosines that recruit phosphotyrosine binding proteins thereby leading to signalling by the ERK1/2, AKT, Src, and p38 MAP kinase pathways. VEGFR1 modulates the activity of VEGFR2, which is the chief pathway in vasculogenesis and angiogenesis. VEGFR3 and its ligands (VEGF-C and VEGF-D) are involved primarily in lymphangiogenesis. Small molecule VEGFR1/2/3 inhibitors including axitinib, cabozantinib, lenvatinib, sorafenib, sunitinib, and pazopanib are approved by the FDA for the treatment of renal cell carcinomas. Most of these agents are type II inhibitors of VEGFR2 and inhibit the so-called DFG-Aspout inactive enzyme conformation. These drugs are steady-state competitive inhibitors with respect to ATP and like ATP they form hydrogen bonds with the hinge residues that connect the small and large protein kinase lobes. Bevacizumab, a monoclonal antibody that binds to VEGF-A, is also approved for the treatment of renal cell carcinomas. Resistance to these agents invariably occurs

  3. Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

    PubMed

    Krastel, Philipp; Roggo, Silvio; Schirle, Markus; Ross, Nathan T; Perruccio, Francesca; Aspesi, Peter; Aust, Thomas; Buntin, Kathrin; Estoppey, David; Liechty, Brigitta; Mapa, Felipa; Memmert, Klaus; Miller, Howard; Pan, Xuewen; Riedl, Ralph; Thibaut, Christian; Thomas, Jason; Wagner, Trixie; Weber, Eric; Xie, Xiaobing; Schmitt, Esther K; Hoepfner, Dominic

    2015-08-24

    Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.

  4. High-resolution genetic mapping of Xa27(t), a new bacterial blight resistance gene in rice, Oryza sativa L.

    PubMed

    Gu, K; Tian, D; Yang, F; Wu, L; Sreekala, C; Wang, D; Wang, G-L; Yin, Z

    2004-03-01

    Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae ( Xoo) (Ishyama) Dye, is one of the serious diseases prevalent throughout Asia. In a previous study, a resistance ( R) locus was transferred from the tetraploid wild rice Oryza minuta to the cultivated rice species, Oryza sativa L. Here, we report the fine genetic mapping of the R locus, tentatively designated as Xa27(t). We performed disease evaluation with an Xa27(t) near-isogenic line, IRBB27, testing 35 Xoo strains collected from 11 countries. The Xa27(t) locus conferred a high level of resistance to 27 strains and moderate resistance to three strains. Resistance of the Xa27(t) gene was developmentally regulated in IRBB27 and showed semi-dominant or a dosage effect in the cv. CO39 genetic background. As a prelude to cloning Xa27(t), a molecular mapping strategy was employed with a large mapping population consisting of 3,875 gametes. Three molecular markers, M336, M1081, and M1059, closely linked to Xa27(t), were identified to facilitate the mapping of Xa27(t) to the long arm of chromosome 6. The Xa27(t) locus was confirmed by chromosome landing of M1081 and M1095 markers on the rice genome. Markers derived from the genomic sequence of O. sativa cv. Nipponbare were used to further saturate the Xa27(t) genomic region. Xa27(t) was finally located within a genetic interval of 0.052 cM, flanked by markers M964 and M1197, and co-segregated with markers M631, M1230, and M449.

  5. Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models.

    PubMed

    Lövgren, K M; Søndergaard, H; Skov, S; Wiinberg, B

    2016-09-01

    In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed.

  6. [Analysis of the Cochrane Review: Direct thrombin inhibitors versus vitamin K antagonists for preventing cerebral or systemic embolism in people with non-valvular atrial fibrillation. Cochrane Database Syst Rev. 2014,3:CD009893].

    PubMed

    Vaz Carneiro, António; Costa, João

    2014-01-01

    Ischemic stroke is one of the most important complications of lone (non-valvular) atrial fibrillation. Its prevention is usually accomplished through oral anticoagulation. Until a few years ago warfarin was the most used agent, but recently two new pharmacologic classes have been introduced for stroke prevention in these patients: oral direct thrombin inhibitors (dabigatran and ximelagatran) and oral factor Xa inhibitors (rivaroxaban, apixaban and edoxaban). In this systematic review, oral direct thrombin inhibitors were compared with warfarin for efficacy and safety. The results indicate that there is no difference in terms of efficacy (except dabigatran 150 mg BID). Oral direct thrombin inhibitors presented less hemorrhages but increased treatment withdrawal due to adverse side-effects (the authors performed post-hoc analyses excluding ximelagatran because this drug was withdrawn from the market owing to safety concerns). There was no difference in terms of mortality between the agents.

  7. Factor VIII alloantibody inhibitors: cost analysis of immune tolerance induction vs. prophylaxis and on-demand with bypass treatment.

    PubMed

    Earnshaw, S R; Graham, C N; McDade, C L; Spears, J B; Kessler, C M

    2015-05-01

    Development of inhibitors (alloantibodies to exogenous factor VIII) is the most significant treatment complication in patients with haemophilia A. The only proven way to eradicate inhibitors is through immune tolerance induction (ITI), while bypassing agents are typically employed to treat or prevent bleeds in patients with high titre inhibitors. Costs of these approaches have not been well studied. The aim of this study was to compare lifetime costs of treating patients with severe haemophilia A with inhibitors using on-demand or prophylaxis treatment with bypassing agents and ITI. A decision-analytic model was developed to compare the treatment costs and outcomes. Quantitation of the reduction in bleeding events for patients on prophylaxis and after eradication of inhibitors when on ITI and relapse of inhibitors was derived from published studies. Costs were obtained from standard US costing sources and are reported in 2014 US dollars. Costs and outcomes were discounted 3% per annum. Lifetime costs of treating patients with inhibitors are lower for ITI vs. on-demand or prophylaxis. Patients are also projected to live longer, have greater quality-adjusted life-years, and have fewer bleeding events than patients treated on-demand. Treating patients via ITI to eradicate inhibitors may result in lower lifetime costs and greater life-years and quality-adjusted life-years than treating with bypassing agents.

  8. Developing an Anti-Xa-Based Anticoagulation Protocol for Patients with Percutaneous Ventricular Assist Devices.

    PubMed

    Sieg, Adam; Mardis, B Andrew; Mardis, Caitlin R; Huber, Michelle R; New, James P; Meadows, Holly B; Cook, Jennifer L; Toole, J Matthew; Uber, Walter E

    2015-01-01

    Because of the complexities associated with anticoagulation in temporary percutaneous ventricular assist device (pVAD) recipients, a lack of standardization exists in their management. This retrospective analysis evaluates current anticoagulation practices at a single center with the aim of identifying an optimal anticoagulation strategy and protocol. Patients were divided into two cohorts based on pVAD implanted (CentriMag (Thoratec; Pleasanton, CA) / TandemHeart (CardiacAssist; Pittsburgh, PA) or Impella (Abiomed, Danvers, MA)), with each group individually analyzed for bleeding and thrombotic complications. Patients in the CentriMag/TandemHeart cohort were subdivided based on the anticoagulation monitoring strategy (activated partial thromboplastin time (aPTT) or antifactor Xa unfractionated heparin (anti-Xa) values). In the CentriMag/TandemHeart cohort, there were five patients with anticoagulation titrated based on anti-Xa values; one patient developed a device thrombosis and a major bleed, whereas another patient experienced major bleeding. Eight patients received an Impella pVAD. Seven total major bleeds in three patients and no thrombotic events were detected. Based on distinct differences between the devices, anti-Xa values, and outcomes, two protocols were created to guide anticoagulation adjustments. However, anticoagulation in patients who require pVAD support is complex with constantly evolving anticoagulation goals. The ideal level of anticoagulation should be individually determined using several coagulation laboratory parameters in concert with hemodynamic changes in the patient's clinical status, the device, and the device cannulation.

  9. Coexpression of heparanase activity, cathepsin L, tissue factor, tissue factor pathway inhibitor, and MMP-9 in proliferative diabetic retinopathy

    PubMed Central

    Siddiquei, Mohammad Mairaj; Nawaz, Mohd Imtiaz; De Hertogh, Gert; Mohammad, Ghulam; Alam, Kaiser; Mousa, Ahmed; Opdenakker, Ghislain

    2016-01-01

    Purpose Heparanase cleaves heparan sulfate side chains of heparan sulfate proteoglycans, activity that is implicated in angiogenesis. Proteolytic cleavage of proheparanase by cathepsin L leads to the formation of catalytically active heparanase. We investigated the expression levels of heparanase enzymatic activity and correlated these with the levels of cathepsin L, the angiogenic factors tissue factor (TF) and matrix metalloproteinase-9 (MMP-9), and the angiostatic factor tissue factor pathway inhibitor (TFPI) in proliferative diabetic retinopathy (PDR). Methods Vitreous samples from 25 patients with PDR and 20 nondiabetic patients and epiretinal membranes from 12 patients with PDR were studied with enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry. Results We observed a significant increase in the expression of heparanase activity in vitreous samples from patients with PDR compared to the nondiabetic controls (p=0.027). Significant positive correlations were found between the levels of heparanase activity and the levels of cathepsin L (r=0.51; p=0.001), TF (r=0.6; p<0.0001), and TFPI (r=0.49; p=0.001). The expression levels of cathepsin L (p=0.019), TF (p<0.0001), TFPI (p<0.0001), and MMP-9 (p=0.029) were significantly higher in the vitreous samples with detected heparanase activity compared to the vitreous samples with undetected heparanase activity. Western blot analysis demonstrated proteolytic cleavage of TFPI in the vitreous samples from patients with PDR. In the epiretinal membranes, cathepsin L, TF, and TFPI were expressed in vascular endothelial cells and CD45-expressing leukocytes. Significant positive correlations were detected between the number of blood vessels that expressed CD31 and the number of blood vessels that expressed TF (r=0.9; p<0.0001) and TFPI (r=0.81; p=0.001). Conclusions The coexpression of these angiogenesis regulatory factors suggests cross-talk between these factors and pathogenesis of PDR

  10. Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage

    PubMed Central

    Bate, Clive; Rumbold, Louis; Williams, Alun

    2007-01-01

    Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902

  11. OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rice Xa21 gene, which confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), encodes a receptor-like kinase. Few components involved in transducing the Xa21-mediated defense response have yet been identified. It is reported that XA21 binds to a WRKY transcription fac...

  12. Inhibition of coagulation proteases Xa and IIa decreases ischemia-reperfusion injuries in a preclinical renal transplantation model.

    PubMed

    Tillet, Solenne; Giraud, Sébastien; Kerforne, Thomas; Saint-Yves, Thibaut; Joffrion, Sandrine; Goujon, Jean-Michel; Cau, Jerôme; Mauco, Gérard; Petitou, Maurice; Hauet, Thierry

    2016-12-01

    Coagulation is an important pathway in the pathophysiology of ischemia-reperfusion injuries. In particular, deceased after circulatory death (DCD) donors undergo a no-flow period, a strong activator of coagulation. Hence, therapies influencing the coagulation cascade must be developed. We evaluated the effect of a new highly specific and effective anti-Xa/IIa molecule, with an integrated innovative antidote site (EP217609), in a porcine preclinical model mimicking injuries observed in DCD donor kidney transplantation. Kidneys were clamped for 60 minutes (warm ischemia), then flushed and preserved for 24 hours at 4°C in University of Wisconsin (UW) solution (supplemented or not). EP217609-supplemented UW solution (UW-EP), compared with unfractionated heparin-supplemented UW solution (UW-UFH) or UW alone (UW). A mechanistic investigation was conducted in vitro: addition of EP217609 to endothelial cells during hypoxia at 4°C in the UW solution inhibited thrombin generation during reoxygenation at 37°C in human plasma and reduced tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 messenger RNA cell expressions. In vivo, function recovery was markedly improved in the UW-EP group. Interestingly, levels of thrombin-antithrombin complexes (reflecting thrombin generation) were reduced 60 minutes after reperfusion in the UW-EP group. In addition, 3 months after transplantation, lower fibrosis, epithelial-mesenchymal transition, inflammation, and leukocyte infiltration were observed. Using this new dual anticoagulant, anti-Xa/IIa activity during kidney flush and preservation is protected by reducing thrombin generation at revascularization, improving early function recovery, and decreasing chronic lesions. Such an easy-to-deploy clinical strategy could improve marginal graft outcome.

  13. KRAS mutational status as a predictor of epidermal growth factor receptor inhibitor efficacy in colorectal cancer.

    PubMed

    Baynes, Roy D; Gansert, Jennifer

    2009-01-01

    Inhibitors of the epidermal growth factor receptor (EGFR) have demonstrated promising potential in the treatment of advanced colorectal cancer. However, a proportion of patients do not respond to therapy with EGFR inhibitors, and therefore, there has been interest in identifying those patients most likely to benefit from therapy with these agents. KRAS, a member of the RAS family of signaling proteins, plays an important role in EGFR-mediated regulation of cellular proliferation and survival. Although there is still some debate regarding the prognostic importance of KRAS mutations in patients with metastatic colorectal cancer, several recent phase 2 and 3 studies have identified the presence of mutations at codons 12 and 13 of KRAS as predictors of poor response to the anti-EGFR monoclonal antibodies panitumumab and cetuximab. Patients with wild-type KRAS were found to have significantly better progression-free survival, overall survival, and/or objective response rate compared with patients harboring KRAS mutations. As a result, there has been growing interest in the development of KRAS mutational status as a biomarker for predicting patient response to EGFR-targeted therapy. Screening colorectal tumors for the absence of KRAS mutations may help identify patients most likely to benefit from anti-EGFR therapies.

  14. Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

    PubMed Central

    Demina, Galina R.; Makarov, Vadim A.; Nikitushkin, Vadim D.; Ryabova, Olga B.; Vostroknutova, Galina N.; Salina, Elena G.; Shleeva, Margarita O.; Goncharenko, Anna V.; Kaprelyants, Arseny S.

    2009-01-01

    Background Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. Methodology/Principal Findings Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. Conclusions/Significance NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins. PMID:20016836

  15. Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells

    PubMed Central

    Spender, Lindsay C.; Ferguson, G. John; Liu, Sijia; Cui, Chao; Girotti, Maria Romina; Sibbet, Gary; Higgs, Ellen B.; Shuttleworth, Morven K.; Hamilton, Tom; Lorigan, Paul; Weller, Michael; Vincent, David F.; Sansom, Owen J.; Frame, Margaret; Dijke, Peter ten; Marais, Richard; Inman, Gareth J.

    2016-01-01

    Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance. PMID:27835901

  16. Discovery of Novel Fibroblast Growth Factor Receptor 1 Kinase Inhibitors by Structure-Based Virtual Screening

    SciTech Connect

    Ravindranathan, K.; Mandiyan, V; Ekkati, A; Bae, J; Schlessinger, J; Jorgensen, W

    2010-01-01

    Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC{sub 50} values of 23 and 50 {micro}M. Initial optimization of 16 led to the more unsaturated 40, which has significantly enhanced potency, 1.9 {micro}M. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

  17. The vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 inhibits aromatase.

    PubMed

    Banerjee, Susana; Zvelebil, Marketa; Furet, Pascal; Mueller-Vieira, Ursula; Evans, Dean B; Dowsett, Mitch; Martin, Lesley-Ann

    2009-06-01

    Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.

  18. Treatment with tumour necrosis factor inhibitor oxpentifylline does not improve corticosteroid dependent chronic active Crohn's disease.

    PubMed Central

    Bauditz, J; Haemling, J; Ortner, M; Lochs, H; Raedler, A; Schreiber, S

    1997-01-01

    BACKGROUND: In Crohn's disease, inflammation is presumably sustained by an increased production of proinflammatory cytokines, in particular tumour necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL 1 beta). TNF alpha can induce a host of cellular effector events resulting in perpetuation of the inflammatory process. In vivo studies with anti-TNF alpha antibody treatment have led to impressive clinical results. AIMS: To investigate whether treatment with the TNF alpha inhibitor oxpentifylline results in clinical improvement in corticosteroid dependent chronic active Crohn's disease. METHODS: Sixteen Crohn's disease patients received oxpentifylline 400 mg four times a day in a four week open label study. RESULTS: Blockade of TNF alpha production in 16 patients with corticosteroid dependent Crohn's disease did not improve the clinical disease activity (CDAI mean (SEM) 188.75 (5.65) versus 185.13 (10.87) or the endoscopic degree of inflammation (CDEIS 14.9 (2.87) versus 14.8 (2.27) or laboratory parameters. CONCLUSIONS: In this study, use of the TNF alpha inhibitor oxpentifylline does not improve inflammation in Crohn's disease. This finding suggests that there may be more key mediators than only TNF alpha in the inflammatory process in Crohn's disease. PMID:9176073

  19. Brunner's gland lesions in rats induced by a vascular endothelial growth factor receptor inhibitor.

    PubMed

    Inomata, Akira; Nakano-Ito, Kyoko; Fujikawa, Yasuhiro; Sonoda, Jiro; Hayakawa, Kazuhiro; Ohta, Etsuko; Taketa, Yoshikazu; Van Gessel, Yvonne; Akare, Sandeep; Hutto, David; Hosokawa, Satoru; Tsukidate, Kazuo

    2014-12-01

    Vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitors are reported to cause reversible mucosal hyperplasia (adenosis) in the duodenum of rats; however, the pathogenesis is not fully elucidated. Using lenvatinib, a VEGF RTK inhibitor, we characterized the histologic time course of this duodenal change in rats. At 4 weeks, there was degeneration and necrosis of Brunner's gland epithelium accompanied by neutrophil infiltration around the affected glands. At 13 weeks, the inflammation was more extensive, and Brunner's gland epithelium was attenuated and flattened and was accompanied by reactive hyperplasia of duodenal epithelium. At 26 weeks, the changes became more severe and chronic and characterized by marked cystic dilation, which extended to the external muscular layer. These dilated glands exhibited morphological characteristics of duodenal crypt epithelium, suggestive of replacement of disappeared Brunner's glands by regenerative duodenal crypt epithelial cells. Similar changes were not present in similar time course studies in dog and monkey studies, suggesting that this is a rodent- or species-specific change. Based on the temporal progression of Brunner's gland lesion, we identify degeneration and necrosis of the Brunner's glands as the primary change leading to inflammation, cystic dilatation, and regeneration with cells that are morphologically suggestive of duodenal crypt epithelium.

  20. Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants.

    PubMed

    Grzesiak, A; Buczek, O; Petry, I; Szewczuk, Z; Otlewski, J

    2000-05-23

    A series of six CMTI I variants mutated in the P(2)-P(4)' region of the canonical binding loop were used to probe the role of single amino acid substitutions on binding to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a). The mutants were expressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escherichia coli, purified from inclusion bodies, followed by cyanobromide cleavage and refolding. The mutants inhibited the proteinases with the association constants in the range 10(3)-10(9) M(-1). Inhibition of plasma kallikrein and factors X(a) and XII(a) could be improved up to 30-fold by single mutations. In contrast, neither of the introduced mutations increased inhibitory properties of CMTI I against plasmin. Additionally, using two inhibitors of natural origin, CMTI I (P(1) Arg) and CPTI II (P(1) Lys), we determined the effect of Lys-->Arg on binding to four proteinases. With the exception of plasmin (no effect), P(1) Arg resulted in up to 30-fold stronger binding than P(1) Lys.

  1. Synthesis and Structure-activity Analysis of Diphenylpyrazolodiazene Inhibitors of the HIV-1 Nef Virulence Factor

    PubMed Central

    Iyer, Prema C.; Zhao, Jielu; Emert-Sedlak, Lori A.; Moore, Kerry; Smithgall, Thomas E.; Day, Billy W.

    2014-01-01

    HIV-1 Nef is a critical AIDS progression factor yet underexplored target for antiretroviral drug discovery. A recent high-throughput screen for pharmacological inhibitors of Nef-dependent Src-family kinase activation identified a diphenylpyrazolodiazene hit compound with submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound, known as ‘B9’, binds directly to Nef and inhibits is dimerization in cells as a possible mechanism of action. Here were synthesized a diverse set of B9 analogs and identified structural features essential to antiretroviral activity. Chemical modifications to each of the three rings present in the parent compound were identified that did not compromise antiviral action. These analogs will guide the development of next-generation compounds with appropriate pharmacological profiles for assessment of antiretroviral activity in vivo. PMID:24650642

  2. QSAR and 3D QSAR of inhibitors of the epidermal growth factor receptor

    NASA Astrophysics Data System (ADS)

    Pinto-Bazurco, Mariano; Tsakovska, Ivanka; Pajeva, Ilza

    This article reports quantitative structure-activity relationships (QSAR) and 3D QSAR models of 134 structurally diverse inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase. Free-Wilson analysis was used to derive the QSAR model. It identified the substituents in aniline, the polycyclic system, and the substituents at the 6- and 7-positions of the polycyclic system as the most important structural features. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used in the 3D QSAR modeling. The steric and electrostatic interactions proved the most important for the inhibitory effect. Both QSAR and 3D QSAR models led to consistent results. On the basis of the statistically significant models, new structures were proposed and their inhibitory activities were predicted.

  3. Delirium in the geriatric unit: proton-pump inhibitors and other risk factors

    PubMed Central

    Otremba, Iwona; Wilczyński, Krzysztof; Szewieczek, Jan

    2016-01-01

    Background Delirium remains a major nosocomial complication of hospitalized elderly. Predictive models for delirium may be useful for identification of high-risk patients for implementation of preventive strategies. Objective Evaluate specific factors for development of delirium in a geriatric ward setting. Methods Prospective cross-sectional study comprised 675 consecutive patients aged 79.2±7.7 years (66% women and 34% men), admitted to the subacute geriatric ward of a multiprofile university hospital after exclusion of 113 patients treated with antipsychotic medication because of behavioral disorders before admission. Comprehensive geriatric assessments including a structured interview, physical examination, geriatric functional assessment, blood sampling, ECG, abdominal ultrasound, chest X-ray, Confusion Assessment Method for diagnosis of delirium, Delirium-O-Meter to assess delirium severity, Richmond Agitation-Sedation Scale to assess sedation or agitation, visual analog scale and Doloplus-2 scale to assess pain level were performed. Results Multivariate logistic regression analysis revealed five independent factors associated with development of delirium in geriatric inpatients: transfer between hospital wards (odds ratio [OR] =2.78; confidence interval [CI] =1.54–5.01; P=0.001), preexisting dementia (OR =2.29; CI =1.44–3.65; P<0.001), previous delirium incidents (OR =2.23; CI =1.47–3.38; P<0.001), previous fall incidents (OR =1.76; CI =1.17–2.64; P=0.006), and use of proton-pump inhibitors (OR =1.67; CI =1.11–2.53; P=0.014). Conclusion Transfer between hospital wards, preexisting dementia, previous delirium incidents, previous fall incidents, and use of proton-pump inhibitors are predictive of development of delirium in the geriatric inpatient setting. PMID:27103793

  4. Synergistic targeting of Sp1, a critical transcription factor for myeloma cell growth and survival, by panobinostat and proteasome inhibitors

    PubMed Central

    Bat-Erdene, Ariunzaya; Miki, Hirokazu; Oda, Asuko; Nakamura, Shingen; Teramachi, Jumpei; Amachi, Ryota; Tenshin, Hirofumi; Hiasa, Masahiro; Iwasa, Masami; Harada, Takeshi; Fujii, Shiro; Sogabe, Kimiko; Kagawa, Kumiko; Yoshida, Sumiko; Endo, Itsuro; Aihara, Kenichi; Abe, Masahiro

    2016-01-01

    Panobinostat, a pan-deacetylase inhibitor, synergistically elicits cytotoxic activity against myeloma (MM) cells in combination with the proteasome inhibitor bortezomib. Because precise mechanisms for panobinostat's anti-MM action still remain elusive, we aimed to clarify the mechanisms of anti-MM effects of panobinostat and its synergism with proteasome inhibitors. Although the transcription factor Sp1 was overexpressed in MM cells, the Sp1 inhibitor terameprocol induced MM cell death in parallel with reduction of IRF4 and cMyc. Panobinostat induced activation of caspase-8, which was inversely correlated with reduction of Sp1 protein levels in MM cells. The panobinostat-mediated effects were further potentiated to effectively induce MM cell death in combination with bortezomib or carfilzomib even at suboptimal concentrations as a single agent. Addition of the caspase-8 inhibitor z-IETD-FMK abolished the Sp1 reduction not only by panobinostat alone but also by its combination with bortezomib, suggesting caspase-8-mediated Sp1 degradation. The synergistic Sp1 reduction markedly suppressed Sp1-driven prosurvival factors, IRF4 and cMyc. Besides, the combinatory treatment reduced HDAC1, another Sp1 target, in MM cells, which may potentiate HDAC inhibition. Collectively, caspase-8-mediated post-translational Sp1 degradation appears to be among major mechanisms for synergistic anti-MM effects of panobinostat and proteasome inhibitors in combination. PMID:27738323

  5. Mechanisms of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance and Strategies to Overcome Resistance in Lung Adenocarcinoma

    PubMed Central

    Chang, Yoon Soo; Choi, Chang-Min

    2016-01-01

    Somatic mutations that lead to hyperactivation of epidermal growth factor receptor (EGFR) signaling are detected in approximately 50% of lung adenocarcinoma in people from the Far East population and tyrosine kinase inhibitors are now the standard first line treatment for advanced disease. They have led to a doubling of progression-free survival and an increase in overall survival by more than 2 years. However, emergence of resistant clones has become the primary cause for treatment failure, and has created a new challenge in the daily management of patients with EGFR mutations. Identification of mechanisms leading to inhibitor resistance has led to new therapeutic modalities, some of which have now been adapted for patients with unsuccessful tyrosine kinase inhibitor treatment. In this review, we describe mechanisms of tyrosine kinase inhibitor resistance and the available strategies to overcoming resistance. PMID:27790276

  6. Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI

    PubMed Central

    Puy, Cristina; Tucker, Erik I.; Ivanov, Ivan S.; Gailani, David; Smith, Stephanie A.; Morrissey, James H.; Gruber, András; McCarty, Owen J. T.

    2016-01-01

    Introduction Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Methods and Results Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Conclusions Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP. PMID:27764259

  7. Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase.

    PubMed

    Yang, Er Bin; Wei, Liu; Zhang, Kai; Chen, Yu Zong; Chen, Wei Ning

    2006-03-01

    Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.

  8. Effect of an epidermal growth factor receptor inhibitor in mouse models of lung cancer.

    PubMed

    Yan, Ying; Lu, Yan; Wang, Min; Vikis, Haris; Yao, Ruisheng; Wang, Yian; Lubet, Ronald A; You, Ming

    2006-12-01

    Gefitinib (Iressa, ZD1839) is a potent high-affinity competitive tyrosine kinase inhibitor aimed primarily at epidermal growth factor receptor (EGFR). Inhibitors in this class have recently been approved for clinical use in the treatment of advanced non-small cell lung cancer as monotherapy following failure of chemotherapy. We examined the efficacy of gefitinib on lung tumorigenesis in mouse models using both postinitiation and progression protocols. Gefitinib was given at a dose of 200 mg/kg body weight (i.g.) beginning either 2 or 12 weeks following carcinogen initiation. In the postinitiation protocol, gefitinib significantly inhibited both tumor multiplicity (approximately 70%) and tumor load (approximately 90%) in A/J or p53-mutant mice (P < 0.0001). Interestingly, gefitinib was also highly effective against lung carcinogenesis in the progression protocol when individual animals already have multiple preinvasive lesions in the lung. Gefitinib exhibited approximately 60% inhibition of tumor multiplicity and approximately 80% inhibition of tumor load when compared with control mice (both P < 0.0001). These data show that gefitinib is a potent chemopreventive agent in both wild-type and p53-mutant mice and that a delayed administration was still highly effective. Analyses of mutations in the EGFR and K-ras genes in lung tumors from either control or treatment groups showed no mutations in EGFR and consistent mutation in K-ras. Using an oligonucleotide array on control and gefitinib-treated lesions showed that gefitinib treatment failed to alter the activity or the expression level of EGFR. In contrast, gefitinib treatment significantly altered the expression of a series of genes involved in cell cycle, cell proliferation, cell transformation, angiogenesis, DNA synthesis, cell migration, immune responses, and apoptosis. Thus, gefitinib showed highly promising chemopreventive and chemotherapeutic activity in this mouse model of lung carcinogenesis.

  9. Captopril attenuates hypertension and renal injury induced by the vascular endothelial growth factor inhibitor sorafenib.

    PubMed

    Nagasawa, Tasuku; Hye Khan, Md Abdul; Imig, John D

    2012-05-01

    Vascular endothelial growth factor inhibitors (VEGFi) are known to cause hypertension and renal injury that severely limits their use as an anticancer therapy. We hypothesized that the angiotensin-converting enzyme inhibitor captopril not only prevents hypertension, but also decreases renal injury caused by the VEGFi sorafenib. Rats were administered sorafenib (20 mg/kg per day) alone or in combination with captopril (40 mg/kg per day) for 4 weeks. Sorafenib administration increased blood pressure, which plateaued by day 10. Concurrent treatment with captopril for 4 weeks resulted in a 30 mmHg decrease in blood pressure compared with sorafenib alone (155 ± 5 vs 182 ± 6 mmHg, respectively; P < 0.05). Furthermore, concurrent captopril treatment reduced albuminuria by 50% compared with sorafenib alone (20 ± 8 vs 42 ± 9 mg/day, respectively; P < 0.05) and reduced nephrinuria by eightfold (280 ± 96 vs 2305 ± 665 μg/day, respectively; P < 0.05). Glomerular injury, thrombotic microangiopathy and tubular cast formation were also decreased in captopril-treated rats administered sorafenib. Renal autoregulatory efficiency was determined by evaluating the afferent arteriolar constrictor response to ATP. Sorafenib administration attenuated the vasoconstriction to ATP, whereas concurrent captopril treatment improved ATP reactivity. In conclusion, captopril attenuated hypertension and renal injury and improved renal autoregulatory capacity in rats administered sorafenib. These findings indicate that captopril treatment, in addition to alleviating the detrimental side-effect of hypertension, decreases the renal injury associated with anticancer VEGFi therapies such as sorafenib.

  10. Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Sutherland, Brian G; Robson, Debra L; Arya, Rigya; Kelly, Karen; Jacobs, René L; Borradaile, Nica M

    2016-09-01

    Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease.

  11. Epidermal growth factor receptor inhibitors trigger a type I interferon response in human skin

    PubMed Central

    Pastore, Saveria

    2016-01-01

    The Epidermal Growth Factor Receptor (EGFR) is centrally involved in the regulation of key processes of the epithelia, including cell proliferation, survival, differentiation, and also tumorigenesis. Humanized antibodies and small-molecule inhibitors targeting EGFR were developed to disrupt these functions in cancer cells and are currently used in the treatment of diverse metastatic epithelial cancers. By contrast, these drugs possess significant skin-specific toxic effects, comprising the establishment of a persistent inflammatory milieu. So far, the molecular mechanisms underlying these epiphenomena have been investigated rather poorly. Here we showed that keratinocytes respond to anti-EGFR drugs with the development of a type I interferon molecular signature. Upregulation of the transcription factor IRF1 is early implicated in the enhanced expression of interferon-kappa, leading to persistent activation of STAT1 and further amplification of downstream interferon-induced genes, including anti-viral effectors and chemokines. When anti-EGFR drugs are associated to TNF-α, whose expression is enhanced by the drugs themselves, all these molecular events undergo a dramatic enhancement by synergy mechanisms. Finally, high levels of interferon-kappa can be observed in epidermal keratinocytes and also in leukocytes infiltrating the upper dermis of cetuximab-driven skin lesions. Our data suggest that dysregulated activation of type I interferon innate immunity is implicated in the molecular processes triggered by anti-EGFR drugs and leading to persistent skin inflammation. PMID:27322144

  12. Design and Synthesis of Novel Small-molecule Inhibitors of the Hypoxia Inducible Factor Pathway

    PubMed Central

    Mooring, Suazette Reid; Jin, Hui; Devi, Narra S.; Jabbar, Adnan A.; Kaluz, Stefan; Liu, Yuan; Van Meir, Erwin G.; Wang, Binghe

    2012-01-01

    Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP. PMID:22032632

  13. Endogenous tissue factor pathway inhibitor has a limited effect on host defence in murine pneumococcal pneumonia.

    PubMed

    van den Boogaard, Florry E; van 't Veer, Cornelis; Roelofs, Joris J T H; Meijers, Joost C M; Schultz, Marcus J; Broze, George J; van der Poll, Tom

    2015-07-01

    Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Coagulation and inflammation interact in the host response to infection. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that inhibits tissue factor (TF), the main activator of inflammation-induced coagulation. It was the objective of this study to investigate the effect of endogenous TFPI levels on coagulation, inflammation and bacterial growth during S. pneumoniae pneumonia in mice. The effect of low endogenous TFPI levels was studied by administration of a neutralising anti-TFPI antibody to wild-type mice, and by using genetically modified mice expressing low levels of TFPI, due to a genetic deletion of the first Kunitz domain of TFPI (TFPIK1(-/-)) rescued with a human TFPI transgene. Pneumonia was induced by intranasal inoculation with S. pneumoniae and samples were obtained at 6, 24 and 48 hours after infection. Anti-TFPI reduced TFPI activity by ~50 %. Homozygous lowTFPI mice and heterozygous controls had ~10 % and ~50 % of normal TFPI activity, respectively. TFPI levels did not influence bacterial growth or dissemination. Whereas lung pathology was unaffected in all groups, mice with ~10 % (but not with ~50 %) of TFPI levels displayed elevated lung cytokine and chemokine concentrations 24 hours after infection. None of the groups with low TFPI levels showed an altered procoagulant response in lungs or plasma during pneumonia. These data argue against an important role for endogenous TFPI in the antibacterial, inflammatory and procoagulant response during pneumococcal pneumonia.

  14. Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis

    PubMed Central

    Rodrigues, Ana Maria; Campanilho-Marques, Raquel; Ponte, Cristina; Canhão, Helena; Ainola, Mari

    2017-01-01

    Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16− monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6. PMID:28286757

  15. Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling.

    PubMed

    Huang, Shiu-Wen; Lien, Jin-Cherng; Kuo, Sheng-Chu; Huang, Tur-Fu

    2012-05-01

    Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.

  16. The potential roles of hepatocyte growth factor (HGF)-MET pathway inhibitors in cancer treatment.

    PubMed

    Parikh, Rahul A; Wang, Peng; Beumer, Jan H; Chu, Edward; Appleman, Leonard J

    2014-01-01

    MET is located on chromosome 7q31 and is a proto-oncogene that encodes for hepatocyte growth factor (HGF) receptor, a member of the receptor tyrosine kinase (RTK) family. HGF, also known as scatter factor (SF), is the only known ligand for MET. MET is a master regulator of cell growth and division (mitogenesis), mobility (motogenesis), and differentiation (morphogenesis); it plays an important role in normal development and tissue regeneration. The HGF-MET axis is frequently dysregulated in cancer by MET gene amplification, translocation, and mutation, or by MET or HGF protein overexpression. MET dysregulation is associated with an increased propensity for metastatic disease and poor overall prognosis across multiple tumor types. Targeting the dysregulated HGF-MET pathway is an area of active research; a number of monoclonal antibodies to HGF and MET, as well as small molecule inhibitors of MET, are under development. This review summarizes the key biological features of the HGF-MET axis, its dysregulation in cancer, and the therapeutic agents targeting the HGF-MET axis, which are in development.

  17. Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study

    PubMed Central

    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Shapiro, Amy D.; Lentz, Steven R.; Wicklund, Brian M.; Kelly, Fiona M.; Soucie, J. Michael

    2015-01-01

    Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)<0.5 and positive on 42/42 specimens (100%) with NBU≥2.0 and 43/80 specimens (53.8%) with NBU 0.5–1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays. PMID:23601690

  18. Intensity of factor VIII treatment and inhibitor development in children with severe hemophilia A: the RODIN study.

    PubMed

    Gouw, Samantha C; van den Berg, H Marijke; Fischer, Kathelijn; Auerswald, Günter; Carcao, Manuel; Chalmers, Elizabeth; Chambost, Hervé; Kurnik, Karin; Liesner, Ri; Petrini, Pia; Platokouki, Helen; Altisent, Carmen; Oldenburg, Johannes; Nolan, Beatrice; Garrido, Rosario Pérez; Mancuso, M Elisa; Rafowicz, Anne; Williams, Mike; Clausen, Niels; Middelburg, Rutger A; Ljung, Rolf; van der Bom, Johanna G

    2013-05-16

    The objective of this study was to examine the association of the intensity of treatment, ranging from high-dose intensive factor VIII (FVIII) treatment to prophylactic treatment, with the inhibitor incidence among previously untreated patients with severe hemophilia A. This cohort study aimed to include consecutive patients with a FVIII activity < 0.01 IU/mL, born between 2000 and 2010, and observed during their first 75 FVIII exposure days. Intensive FVIII treatment of hemorrhages or surgery at the start of treatment was associated with an increased inhibitor risk (adjusted hazard ratio [aHR], 2.0; 95% confidence interval [CI], 1.3-3.0). High-dose FVIII treatment was associated with a higher inhibitor risk than low-dose FVIII treatment (aHR, 2.3; 95% CI, 1.0-4.8). Prophylaxis was only associated with a decreased overall inhibitor incidence after 20 exposure days of FVIII. The association with prophylaxis was more pronounced in patients with low-risk F8 genotypes than in patients with high-risk F8 genotypes (aHR, 0.61, 95% CI, 0.19-2.0 and aHR, 0.85, 95% CI, 0.51-1.4, respectively). In conclusion, our findings suggest that in previously untreated patients with severe hemophilia A, high-dosed intensive FVIII treatment increases inhibitor risk and prophylactic FVIII treatment decreases inhibitor risk, especially in patients with low-risk F8 mutations.

  19. A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors

    PubMed Central

    2016-01-01

    Human macrophage migration inhibitory factor (MIF) is both a keto–enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein–ligand hydrogen bonding, aryl–aryl, and cation−π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders. PMID:27299179

  20. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    PubMed Central

    Kang, Minyong; Lee, Kyoung-Hwa; Lee, Hye Sun; Jeong, Chang Wook; Kwak, Cheol; Kim, Hyeon Hoe; Ku, Ja Hyeon

    2017-01-01

    Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel

  1. 76 FR 80829 - Special Conditions: XtremeAir GmbH, XA42; Acrobatic Category Aerodynamic Stability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-27

    ... airplane. The XA42 airplane has a novel or unusual design feature associated with its static stability... acrobatic capability: Neutral longitudinal and lateral static stability characteristics. Discussion The Code of Federal Regulations states static stability criteria for longitudinal, lateral, and...

  2. Acoustic Emission Monitoring of the DC-XA Composite Liquid Hydrogen Tank During Structural Testing

    NASA Technical Reports Server (NTRS)

    Wilkerson, C.

    1996-01-01

    The results of acoustic emission (AE) monitoring of the DC-XA composite liquid hydrogen tank are presented in this report. The tank was subjected to pressurization, tensile, and compressive loads at ambient temperatures and also while full of liquid nitrogen. The tank was also pressurized with liquid hydrogen. AE was used to monitor the tank for signs of structural defects developing during the test.

  3. The First Test Flight of the Delta Clipper-Experimental Advanced (DC-XA)

    NASA Technical Reports Server (NTRS)

    1966-01-01

    The Delta Clipper-Experimental Advanced (DC-XA) is a single-stage-to-orbit, vertical takeoff / vertical landing launch vehicle concept, whose development was geared to significantly reduce launch cost and provided a test bed for NASA Reusable Launch Vehicle (RLV) technology. This photograph shows the descending vehicle landing during the first successful test flight at White Sands Missile Range, New Mexico. The program was discontinued in 2003.

  4. Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

    PubMed Central

    Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

    2014-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

  5. Induction of an inhibitor antibody to factor XI in a patient with severe inherited factor XI deficiency by Rh immune globulin.

    PubMed

    Zucker, Michal; Zivelin, Ariella; Teitel, Jerome; Seligsohn, Uri

    2008-02-01

    In this paper, we report an inhibitor antibody to factor XI (FXI) in a woman with severe inherited FXI deficiency, induced by FXI present in an Rh immune globulin preparation. The patient is homozygous for the Glu117Stop mutation, associated with a FXI level of less than 1 U/dL. Unlike all previously described patients with severe FXI deficiency and an inhibitor, the patient had never been exposed to blood products. Following 3 injections of Rh immune globulin during pregnancy, she developed an inhibitor to FXI (8 Bethesda units) that was shown to bind specifically to FXI and inhibit factor IX cleavage by purified FXIa. The administered Rh immune globulin and 2 other similar products were shown to contain FXI. Clinicians should be aware of the potential for immunization of severely FXI-deficient patients by FXI present in Rh immune globulin preparations.

  6. Comparative nutritional compositions and proteomics analysis of transgenic Xa21 rice seeds compared to conventional rice.

    PubMed

    Gayen, Dipak; Paul, Soumitra; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2016-07-15

    Transgenic rice expressing the Xa21 gene have enhanced resistant to most devastating bacterial blight diseases caused by Xanthomonas oryzae pv. oryzae (Xoo). However, identification of unintended modifications, owing to the genetic modification, is an important aspect of transgenic crop safety assessment. In this study, the nutritional compositions of seeds from transgenic rice plants expressing the Xa21 gene were compared against non-transgenic rice seeds. In addition, to detect any changes in protein translation levels as a result of Xa21 gene expression, rice seed proteome analyses were also performed by two-dimensional gel electrophoresis. No significant differences were found in the nutritional compositions (proximate components, amino acids, minerals, vitamins and anti-nutrients) of the transgenic and non-transgenic rice seeds. Although gel electrophoresis identified 11 proteins that were differentially expressed between the transgenic and non-transgenic seed, only one of these (with a 20-fold up-regulation in the transgenic seed) shows nutrient reservoir activity. No new toxins or allergens were detected in the transgenic seeds.

  7. Materials Testing on the DC-X and DC-XA

    NASA Technical Reports Server (NTRS)

    Smith, Dane; Carroll, Carol; Marschall, Jochen; Pallix, Joan

    1997-01-01

    Flight testing of thermal protection materials has been carried out over a two year period on the base heat shield of the Delta Clipper (DC-X and DC-XA), as well on a body flap. The purpose was to use the vehicle as a test bed for materials and more efficient repair or maintenance processes which would be potentially useful for application on new entry vehicles (i.e., X-33, RLV, planetary probes), as well as on the existing space shuttle orbiters. Panels containing Thermal Protection Systems (TPS) and/or structural materials were constructed either at NASA Ames Research Center or at McDonnell Douglas Aerospace (MDA) and attached between two of the four thrusters in the base heat shield of the DC-X or DC-XA. Three different panels were flown on DC-X flights 6, 7, and 8. A total of 7 panels were flown on DC-XA flights 1, 2, and 3. The panels constructed at Ames contained a variety of ceramic TPS including flexible blankets, tiles with high emissivity coatings, lightweight ceramic ablators and other ceramic composites. The MDS test panels consisted primarily of a variety of metallic composites. This report focuses on the ceramic TPS test results.

  8. Characteristics of hemophilia patients with factor VIII inhibitors detected by prospective screening

    PubMed Central

    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Payne, Amanda B.; Kelly, Fiona M.; Escobar, Miguel A.; Gill, Joan; Leissinger, Cindy; Soucie, J. Michael

    2015-01-01

    Characteristics of inhibitors identified by prospective screening may differ from those detected clinically. In a prospective study at 17 hemophilia centers with central inhibitor measurement by Nijmegen-Bethesda assay, 23 (2.8%) of 824 hemophilia A patients had new inhibitors detected: nine high-titer inhibitors (HTI: 7 ≥5.0 NBU plus 2 of 2.6 and 3.4 NBU at immune tolerance induction initiation) and 14 low-titer inhibitors (LTI: 0.5–1.9 NBU). HTI occurred at an earlier age (median 2 years, range 1–18, vs. median 11 years, range 2–61, P = 0.016). Both HTI (22%) and LTI (43%) occurred in non-severe patients. All HTI, but only 64% of LTI, were found to be FVIII-specific by chromogenic Bethesda assay or fluorescence immunoassay (FLI), indicating a high rate of false-positive LTI. Repeat specimens confirmed all HTI, 7/9 LTI, and 7/7 FVIII-specific LTI. FLI results were similar between HTI and FVIII-specific LTI; all included IgG1 and IgG4 subclasses. A comparable prospective study conducted from 1975 to 1979 at 13 U.S. centers found 31 (2.4%) new inhibitors among 1,306 patients. In both studies, one-third of inhibitors occurred in non-severe patients and one-quarter after 150 exposure days (ED). Significant differences were seen in the age at which inhibitors occurred (median 16 years in the older study vs. 5 years currently, P = 0.024) and in ED before inhibitor development, 10% in the older study and 43% currently study occurring within 20 ED, suggesting a temporal change in inhibitor development. Prospective screening detects inhibitors in patients of all severities, ages, and ED. Some LTI, however, are false positives. PMID:26147783

  9. Effects of tumor necrosis factor α inhibitors extend beyond psoriasis: insulin sensitivity in psoriasis patients with type 2 diabetes mellitus.

    PubMed

    Al-Mutairi, Nawaf; Shabaan, Dalia

    2016-03-01

    Psoriasis is a chronic inflammatory disease that has been associated with an increased incidence of insulin resistance and diabetes mellitus (DM). Tumor necrosis factor (TNF) α inhibitors and IL-6 blockers, which are routinely used for the treatment of psoriasis, have been positively associated with insulin sensitivity. The aim of this study was to assess the effects of treatment with TNF-α inhibitors on insulin sensitivity in psoriatic patients with type 2 DM. This study confirms a beneficial effect of anti-TNF-α agents on insulin resistance and insulin sensitivity in psoriasis patients with type 2 DM.

  10. Regulation of epidermal growth factor receptor signalling by inducible feedback inhibitors.

    PubMed

    Segatto, Oreste; Anastasi, Sergio; Alemà, Stefano

    2011-06-01

    Signalling by the epidermal growth factor receptor (EGFR) controls morphogenesis and/or homeostasis of several tissues from worms to mammals. The correct execution of these programmes requires the generation of EGFR signals of appropriate strength and duration. This is obtained through a complex circuitry of positive and negative feedback regulation. Feedback inhibitory mechanisms restrain EGFR activity in time and space, which is key to ensuring that receptor outputs are commensurate to the cell and tissue needs. Here, we focus on the emerging field of inducible negative feedback regulation of the EGFR in mammals. In mammalian cells, four EGFR inducible feedback inhibitors (IFIs), namely LRIG1, RALT (also known as MIG6 and ERRFI1), SOCS4 and SOCS5, have been discovered recently. EGFR IFIs are expressed de novo in the context of early or delayed transcriptional responses triggered by EGFR activation. They all bind to the EGFR and suppress receptor signalling through several mechanisms, including catalytic inhibition and receptor downregulation. Here, we review the mechanistic basis of IFI signalling and rationalise the function of IFIs in light of gene-knockout studies that assign LRIG1 and RALT an essential role in restricting cell proliferation. Finally, we discuss how IFIs might participate in system control of EGFR signalling and highlight the emerging roles for IFIs in the suppression of EGFR-driven tumorigenesis.

  11. Tumor necrosis factorinhibitor therapy and fetal risk: A systematic literature review

    PubMed Central

    Marchioni, Renée M; Lichtenstein, Gary R

    2013-01-01

    Tumor necrosis factorinhibitors (anti-TNFs) are effective in the treatment of inflammatory bowel disease (IBD) recalcitrant to conventional medical therapy. As the peak incidence of IBD overlaps with the prime reproductive years, it is crucial to establish pharmacologic regimens for women of childbearing age that achieve effective disease control without posing significant fetal harm. A systematic literature review was performed to identify all human studies with birth outcomes data after maternal exposure to infliximab, adalimumab, or certolizumab pegol within 3 mo of conception or during any trimester of pregnancy. Live births, spontaneous abortions or stillbirths, preterm or premature births, low birth weight or small for gestational age infants, and congenital abnormalities were recorded. Fifty selected references identified 472 pregnancy exposures. The subsequent review includes general information regarding anti-TNF therapy in pregnancy followed by a summary of our findings. The benefits of biologic modalities in optimizing disease control during pregnancy must be weighed against the potential toxicity of drug exposure on the developing fetus. Although promising overall, there is insufficient evidence to prove absolute safety for use of anti-TNFs during pregnancy given the limitations of available data and lack of controlled trials. PMID:23674866

  12. A-62176, a potent topoisomerase inhibitor, inhibits the expression of human epidermal growth factor receptor 2.

    PubMed

    Kim, Hye-Lin; Jeon, Kyung-Hwa; Jun, Kyu-Yeon; Choi, Yongmun; Kim, Dae-Kee; Na, Younghwa; Kwon, Youngjoo

    2012-12-01

    HER2 overexpression is observed in ∼6-35% of all gastric cancers, while co-amplification of topoisomerase IIα occurs in ∼32-90% of all cancers with HER2 amplification. The present study reports that HER2 expression is down-regulated by A-62176, a fluoroquinophenoxazine derivative that we previously demonstrated to inhibit topoisomerase I and IIα. The results suggest that A-62176 inhibits the interaction between the ESX, an ets transcription factor, and its co-activator Sur2, leading to the attenuation of HER2-mediated phosphorylation of MAPK/Akt. A-62176 arrests the cell cycle in the G1 phase via the down-regulation of cyclin D1 and the up-regulation of p27(Kip1) in NCI-N87 gastric cancer cells. The combination of A-62176 with doxorubicin provides a strong synergistic activity. We propose that A-62176 is a dual inhibitor that impairs the expression of HER2 and restrains the activity of topoisomerase IIα. Our results may lead to the rational design of anticancer molecules targeting a subgroup of gastric cancer cells overexpressing both HER2 and topoisomerase IIα.

  13. Addressing epidermal growth factor receptor tyrosine kinase inhibitor resistance in non-small cell lung cancer.

    PubMed

    Noda, Shoko; Kanda, Shintaro

    2016-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have significantly improved the survival of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. However, nearly all EGFR-mutant NSCLC tumors eventually acquire resistance to the currently used EGFR-TKIs and subsequently progress clinically. Acquired resistance to EGFR-TKIs is thus a huge issue in the treatment of EGFR-mutant NSCLC at present. On one hand, T790M second-site mutation has been recognized as a key mechanism of EGFR-TKI resistance, and third generation EGFR-TKIs such as osimertinib and rociletinib have been developed to overcome tumor cells harboring the T790M mutation. On the other hand, combination with cytotoxic chemotherapy is also expected as another strategy for preventing the acquired resistance to current EGFR-TKIs and prolonging the survival benefits by EGFR-TKIs. Here, we review updated strategies for preventing or overcoming acquired resistance to EGFR-TKIs.

  14. Epidermal growth factor receptor inhibitor protects against abdominal aortic aneurysm in a mouse model.

    PubMed

    Obama, Takashi; Tsuji, Toshiyuki; Kobayashi, Tomonori; Fukuda, Yamato; Takayanagi, Takehiko; Taro, Yoshinori; Kawai, Tatsuo; Forrester, Steven J; Elliott, Katherine J; Choi, Eric; Daugherty, Alan; Rizzo, Victor; Eguchi, Satoru

    2015-05-01

    Angiotensin II (Ang II) has been implicated in the development of abdominal aortic aneurysm (AAA). In vascular smooth muscle cells (VSMC), Ang II activates epidermal growth factor receptor (EGFR) mediating growth promotion. We hypothesized that inhibition of EGFR prevents Ang II-dependent AAA. C57BL/6 mice were co-treated with Ang II and β-aminopropionitrile (BAPN) to induce AAA with or without treatment with EGFR inhibitor, erlotinib. Without erlotinib, 64.3% of mice were dead due to aortic rupture. All surviving mice had AAA associated with EGFR activation. Erlotinib-treated mice did not die and developed far fewer AAA. The maximum diameters of abdominal aortas were significantly shorter with erlotinib treatment. In contrast, both erlotinib-treated and non-treated mice developed hypertension. The erlotinib treatment of abdominal aorta was associated with lack of EGFR activation, endoplasmic reticulum (ER) stress, oxidative stress, interleukin-6 induction and matrix deposition. EGFR activation in AAA was also observed in humans. In conclusion, EGFR inhibition appears to protect mice from AAA formation induced by Ang II plus BAPN. The mechanism seems to involve suppression of vascular EGFR and ER stress.

  15. Hemofiltration during cardiopulmonary bypass: the effect on anti-Xa and anti-IIa heparin activity.

    PubMed

    Despotis, G J; Levine, V; Filos, K S; Joiner-Maier, D; Joist, J H

    1997-03-01

    Previous studies have demonstrated that heparin concentrations during cardiopulmonary bypass (CPB) may vary considerably, which may be related to variability in redistribution, cellular and plasma protein binding, and clearance of heparin. The purpose of this study was to determine whether hemofiltration removes lower molecular weight fractions of heparin from plasma and thus contributes to variability of blood levels of heparin. Twenty patients undergoing cardiac surgery with CPB were enrolled in this study after informed consent was obtained. The study was subdivided into two phases. The first 10 patients were enrolled in Phase I, which was designed to determine whether hemofiltration removes lower molecular weight fractions of heparin from blood. Blood specimens obtained from the inflow line and outflow lines of the hemofiltration unit were used to measure complete blood counts (CBC) and plasma heparin activity by anti-Xa and anti-IIa assays. Phase II was designed to evaluate the effect of hemofiltration on circulating plasma heparin activity. In Phase II, blood specimens were obtained from 10 patients via the arterial cannula of the extracorporeal circuit prior to and after hemofiltration for measurements of CBCs, anti-Xa plasma heparin, as well as whole blood heparin concentration using an automated protamine titration assay (Hepcon instrument, Medtronic Inc., Parker, CO). Ultrafiltrate and reservoir volumes were measured in both phases of the study. Hemofiltration did not remove lower (anti-Xa measurable) molecular weight heparin, but it resulted in a considerable increase in heparin activity in the outflow line, as measured by both anti-Xa and anti-IIa assays. The plasma anti-Xa heparin activity obtained after hemofiltration (5 +/- 1.8 U/mL) was substantially (P = 0.003) greater than heparin activity obtained prior to hemofiltration (3.9 +/- 1.7 U/mL). The increase in heparin activity with hemofiltration was directly related to ultrafiltrate volume (r = 0

  16. Factors associated with residual gastroesophageal reflux disease symptoms in patients receiving proton pump inhibitor maintenance therapy

    PubMed Central

    Kawara, Fumiaki; Fujita, Tsuyoshi; Morita, Yoshinori; Uda, Atsushi; Masuda, Atsuhiro; Saito, Masaya; Ooi, Makoto; Ishida, Tsukasa; Kondo, Yasuyuki; Yoshida, Shiei; Okuno, Tatsuya; Yano, Yoshihiko; Yoshida, Masaru; Kutsumi, Hiromu; Hayakumo, Takanobu; Yamashita, Kazuhiko; Hirano, Takeshi; Hirai, Midori; Azuma, Takeshi

    2017-01-01

    AIM To elucidate the factors associated with residual gastroesophageal reflux disease (GERD) symptoms in patients receiving proton pump inhibitor (PPI) maintenance therapy in clinical practice. METHODS The study included 39 GERD patients receiving maintenance PPI therapy. Residual symptoms were assessed using the Frequency Scale for Symptoms of GERD (FSSG) questionnaire and the Gastrointestinal Symptom Rating Scale (GSRS). The relationships between the FSSG score and patient background factors, including the CYP2C19 genotype, were analyzed. RESULTS The FSSG scores ranged from 1 to 28 points (median score: 7.5 points), and 19 patients (48.7%) had a score of 8 points or more. The patients’ GSRS scores were significantly correlated with their FSSG scores (correlation coefficient = 0.47, P < 0.005). In erosive esophagitis patients, the FSSG scores of the CYP2C19 rapid metabolizers (RMs) were significantly higher than the scores of the poor metabolizers and intermediate metabolizers (total scores: 16.7 ± 8.6 vs 7.8 ± 5.4, P < 0.05; acid reflux-related symptom scores: 12 ± 1.9 vs 2.5 ± 0.8, P < 0.005). In contrast, the FSSG scores of the CYP2C19 RMs in the non-erosive reflux disease patients were significantly lower than those of the other patients (total scores: 5.5 ± 1.0 vs 11.8 ± 6.3, P < 0.05; dysmotility symptom-related scores: 1.0 ± 0.4 vs 6.0 ± 0.8, P < 0.01). CONCLUSION Approximately half of the GERD patients receiving maintenance PPI therapy had residual symptoms associated with a lower quality of life, and the CYP2C19 genotype appeared to be associated with these residual symptoms. PMID:28373773

  17. Predictive Factors of Response to Proton Pump Inhibitors in Korean Patients With Gastroesophageal Reflux Disease

    PubMed Central

    Kim, Sung Eun; Kim, Nayoung; Oh, Sooyeon; Kim, Hee Man; Park, Moo In; Lee, Dong Ho; Jung, Hyun Chae

    2015-01-01

    Background/Aims Proton pump inhibitors (PPIs) are widely used in the treatment of gastroesophageal reflux disease (GERD). However, some patients fail to respond to PPI therapy. We investigated the efficacy of response to PPI therapy in patients with GERD symptoms. Methods A total of 179 subjects with GERD symptoms were prospectively enrolled and diagnosed with non-erosive reflux disease (NERD, n = 100) and erosive reflux disease (n = 79) by gastroscopy and Bernstein test and/or 24-hour esophageal pH testing. Subjects then received a standard dose of daily PPI therapy for at least 4 weeks. PPI therapy response was evaluated using questionnaires including questions about demographics, GERD symptoms, GERD impact scale, Epworth sleepiness scale, Pittsburgh sleep quality index (PSQI), hospital anxiety and depression scale, and abbreviated version of the World Health Organization quality of life scale. Results The rates of complete (≥ 80%), satisfactory (≥ 50%), partial (< 50%), and refractory response in the 179 participants were 41.3%, 30.2%, 18.4%, and 10.1%, respectively. Thus, overall response rate (complete and satisfactory responses) was 71.5%. Multivariate analysis showed body mass index < 23 kg/m2 (OR, 2.20; 95% CI, 1.12–4.34), higher total PSQI score (OR, 1.20; 95% CI, 1.05–1.35), history of psychotherapy or neuropsychiatric medication (OR, 2.44; 95% CI, 1.23–4.85), and NERD (OR, 3.30; 95% CI, 1.54–7.11) were associated with poor response to PPI therapy. Conclusions Psychological factors, sleep dysfunction, body mass index < 23 kg/m2, and NERD seem to be the major factors that lead to a poor response to PPI treatment in patients with GERD symptoms. PMID:25537676

  18. The M358R variant of α(1)-proteinase inhibitor inhibits coagulation factor VIIa.

    PubMed

    Sheffield, William P; Bhakta, Varsha

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg-Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg-Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10(2) M(-1)sec(-1). We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin.

  19. Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro.

    PubMed Central

    Papapetropoulos, A.; Desai, K. M.; Rudic, R. D.; Mayer, B.; Zhang, R.; Ruiz-Torres, M. P.; García-Cardeña, G.; Madri, J. A.; Sessa, W. C.

    1997-01-01

    Angiogenesis is a complex process involving endothelial cell (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-beta 1. Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-beta 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs. Images Figure 2 Figure 4 Figure 5 PMID:9137106

  20. Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Ankylosing Spondylitis

    PubMed Central

    Caetano-Lopes, Joana; Vieira-Sousa, Elsa; Campanilho-Marques, Raquel; Ponte, Cristina; Canhão, Helena; Ainola, Mari; Fonseca, João E.

    2015-01-01

    Introduction Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. Methods 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. Results RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-β levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. Conclusion In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli. PMID:26674064

  1. The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin.

    PubMed

    Al Otair, Hadil A; Abdel Gader, Abdel Galil M; Khurshid, Syed M; Alzeer, Abdulaziz H; Al Momen, Abdul Kareem; Al Shaikh, Mashael; Al Gahtani, Farja; Al Aseri, Zohair A; Abdelrazik, Hossam A H

    2016-06-05

    Amaç: Sepsis sendromuna genellikle kan pıhtılaşma sisteminin aktivasyonu eşlik eder. İlk çalışmalar ana doğal 3 antikoagülan olan antitrombin, protein C ve protein S eksikliği bulmuştur. Bununla birlikte, bu inhibitörlerin hiç biri doku faktörü bloke etmez, sepsis sırasındaki koagülasyon tetiklenişi özelllikle doku faktör yolak inhibitörü (DFYİ) ile kontrol edilir. Bu çalışmanın amacı sepsis sırasındaki doğal antikoagülan ve özellikle DFYİ düzeyi dalgalanmalarını karakterize etmek ve bunların düşük moleküler ağırlıklı heaprin enoksaparinin antikoagülan eylemi ile ilişkilerini öğrenmekti. Gereç ve Yöntemler: Ardışık 51 sepsis hastası çalışmaya alındı. Taban (0 saat) ve enoksaparin verimesinden 4, 12, 24 saat sonra kan örnekleri alındı. Aşağıdaki deneyler ticari kitleri kullanılarak yapılmıştır; parsiyel tromboplastin zamanı, protrombin zamanı, trombin zamanı, toplam ve serbest DFYİ, protein C ve protein S, antitrombin, fibrinojen, ve aktif anti-faktör Xa. Bulgular: Enoksaparin uygulamadan önce ptorombin zamanı ve aktif parsiyel protrombin zamanında önemli uzama vardı. Bu durum sonraki 3 örneklemde de devam etti. Çalışma boyunca antitrombin, protein C, toplam ve serbest protein S seviyeleri değerlerinde kontrollere göre belirgin bir azalma oldu. Buna karşılık, hem toplam hem de serbest plazma DFYİ değerleri belirgin biçimde yükseldi ve enoksaparin tedavisinden sonra arttı. Anti faktör Xa düzeyleri terapötik aralık içindeydi. Vefat eden ve sağ kalan hastalar arasında DFYİ düzeyi açısından fark yoktu. Sonuç: Sepsis, endotel hücrelerinden belirgin DFYİ salınımı ile tetiklenir. Bu, enoksaparin uygulmasını takiben kalıcı olmuş ve daha da artmıştır. Bunun aksine, doğal koagülasyon inhibitörleri antitrombin, protein C ve protein S’nin belirgin tüketimi vardı. Bu sonuçlar, tedavi amaçlı rekombinant DFYİ kullanımının sepsis ilişkili koag

  2. Hemophilia as a defect of the tissue factor pathway of blood coagulation: Effect of factors VIII and IX on factor X activation in a continuous-flow reactor

    SciTech Connect

    Repke, D.; Gemmell, C.H.; Guha, A.; Turitto, V.T.; Nemerson, Y. ); Broze, G.J. Jr. )

    1990-10-01

    The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a wall shear rate of 57, 300, or 1130 sec{sup {minus}1}. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2{endash}-to 3{endash}fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.

  3. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

    PubMed Central

    Vuimo, Tatiana A.; Surov, Stepan S.; Ovsepyan, Ruzanna A.; Korneeva, Vera A.; Vorobiev, Ivan I.; Orlova, Nadezhda A.; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V.; Ataullakhanov, Fazoil I.; Panteleev, Mikhail A.

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  4. Acquired factor V inhibitor in a patient receiving venous-venous extracorporeal membrane oxygenation for Legionella pneumonia.

    PubMed

    Leung, Anne K H; Ng, George W Y; Sin, K C; Au, S Y; Lai, K Y; Lee, K L; Law, K I

    2015-04-01

    We report a rare complication of factor V deficiency in a patient having Legionella pneumonia. This patient also had other complications like severe acute respiratory distress syndrome, acute kidney injury, and septic shock that required venous-venous extracorporeal membrane oxygenation support. This is the first reported case of acquired factor V deficiency in a patient receiving extracorporeal membrane oxygenation for Legionella pneumonia. With the combined use of intravenous immunoglobulin, rituximab and plasma exchange, we achieved rapid clearance of the factor V inhibitor within 1 week so as to allow safe decannulation of extracorporeal membrane oxygenation.

  5. Tissue factor-driven thrombin generation and inflammation in atherosclerosis.

    PubMed

    ten Cate, Hugo

    2012-05-01

    The transmembrane receptor tissue factor is a prominent protein expressed at macrophages and smooth muscle cells within human atherosclerotic lesions. While many coagulation proteins are detectable in atherosclerosis, a locally active thrombin and fibrin generating molecular machinery may be instrumental in manipulating cellular functions involved in atherogenesis. These include inflammation, angiogenesis and cell proliferation. Indeed, many experimental studies in mice show a correlation between hypercoagulability and increased atherosclerosis. In mice, the amount of atherosclerosis and/or the plaque phenotype, appear to be modifiable by specific anticoagulant interventions. While attempts to vary tissue factor level in the vasculature does not directly reduce plaque burden, the overexpression of tissue factor pathway inhibitor attenuates thrombogenicity and neo intima formation in mice. Moreover, inhibition of factor Xa or thrombin with novel selective agents, including rivaroxaban and dabigatran, inhibits inflammation associated with atherosclerosis in apoE(-/-) mice. The potential to modify a complex chronic disease like atherosclerosis with novel selective anticoagulants merits further clinical study.

  6. Predictive factors of renal toxicities related to anti-VEGFR multikinase inhibitors in phase 1 trials.

    PubMed

    Boissier, Emilie; Mir, Olivier; Hollebecque, Antoine; Izzedine, Hassan; Ederhy, Stéphane; Gazzah, Anas; Bahleda, Rastislav; Massard, Christophe; Macquin-Mavier, Isabelle; Tournigand, Christophe; Spano, Jean-Philippe; Soria, Jean-Charles; Rousseau, Benoît

    2017-02-01

    Purpose Renal toxicities are common with angiogenesis multikinase inhibitors (AMKI), and can be limiting in phase I trials. Factors associated with such toxicities are poorly known. The aims of this exploratory study were to describe renovascular toxicities associated with AMKI, impact on drug development and to identify baseline parameters associated with the occurrence of renal toxicities in phase I trials. Methods Consecutive patients treated with AMKI in Gustave Roussy phase I unit between October 2005 and August 2013 were included. We retrospectively collected baseline characteristics and renovascular side effects. Associations were assessed in univariate and multivariate analyses. Results Overall, 168 patients were included: male 53.0 %, mean age 55.5 years old, history of hypertension 26.8 %, diabetes 6.0 %, atherosclerosis 13.6 %, stage 3 Chronic Kidney Disease (CKD, NKF-KDOQI) 17.2 %. Incidences of reno-vascular side effects were: hypertension 47.6 %, proteinuria 19.0 %, renal failure 11.9 % and thrombotic microangiopathy 10.1 %. Eighty percent of dose limiting toxicities (DLTs) were related to a renal toxicity. Multivariate analysis showed that onset of renal failure was associated with history of hypertension (p = 0.0003) and stage 3 CKD (p = 0.032). Conclusions A majority of the DLTs associated with AMKI in phase 1 trials are renal toxicities. Baseline hypertension and stage 3 CKD (NKF-KDOQI) might help to better identify patients at risk of AMKI-related renal toxicities.

  7. Impact of Dose Tapering of Tumor Necrosis Factor Inhibitor on Radiographic Progression in Ankylosing Spondylitis

    PubMed Central

    Park, Jun Won; Kwon, Hyun Mi; Park, Jin Kyun; Choi, Ja-Young; Lee, Eun Bong; Song, Yeong Wook

    2016-01-01

    Objective To investigate the impact of dose reduction of tumor necrosis factor inhibitor (TNFi) on radiographic progression in ankylosing spondylitis (AS). Methods One hundred and sixty-five patients treated with etanercept or adalimumab were selected from a consecutive single-center observational cohort based on the availability of radiographs at baseline and after two- and/or four-years of follow up. Radiographs were assessed by two blinded readers using the modified Stokes AS Spinal Score (mSASSS). Radiographic progression in patients treated with standard-dose TNFi (standard-dose group, n = 49) was compared with patients whose dosage was tapered during the treatment (tapering group, n = 116) using linear mixed models. Results Baseline characteristics between two groups were comparable except for higher BASDAI (7.1 vs. 6.3, p = 0.003) in the standard-dose group. At two years after the treatment, mean dose quotient (S.D.) of the tapering group was 0.59 (0.17). During follow up, rate of radiographic progression in overall patients was 0.90 mSASSS units/year. Radiographic progression over time between the two groups was similar at the entire group level. However, in the subgroup of patients with baseline syndesmophytes, progression occurred significantly faster in the tapering group after the adjustment for baseline status (1.23 vs. 1.72 mSASSS units/year, p = 0.023). Results were consistent when radiographic progression was assessed by the number of newly developed syndesmophytes (0.52 vs. 0.73/year, p = 0.047). Sensitivity analysis after multiple imputation of missing radiographs also showed similar results. Conclusion A dose tapering strategy of TNFi is associated with more rapid radiographic progression in AS patients who have syndesmophytes at baseline. PMID:28033420

  8. Polymorphisms in antithrombin and in tissue factor pathway inhibitor genes are associated with recurrent pregnancy loss.

    PubMed

    Guerra-Shinohara, Elvira M; Bertinato, Juliano Felix; Tosin Bueno, Carolina; Cordeiro da Silva, Kelma; Burlacchini de Carvalho, Mário Henrique; Pulcineli Vieira Francisco, Rossana; Zugaib, Marcelo; Cerda, Alvaro; Morelli, Vânia Maris

    2012-10-01

    Recurrent pregnancy loss (RPL) is a multifactorial condition. The effect of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) single nucleotide polymorphisms (SNPs) on the risk of RPL is thus far unknown. Our objective was to determine the association of SNPs in the above mentioned genes with RPL. We included 117 non-pregnant women with three or more consecutive losses prior to 20 weeks of pregnancy without a previous history of carrying a fetus to viability, and 264 healthy fertile non-pregnant women who had at least two term deliveries and no known pregnancy losses. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292, rs8176592 and rs10153820) SNPs were analysed by Real Time PCR. Genotype frequencies for PROC 2418A>G, PROC 2405C>T, THBD 1418C>T, TFPI (T-33C and TFPI C-399T) SNPs were similar in cases and controls. The carriers of SERPINC1 786A allele (GA + AA genotypes) had an increased risk for RPL (odds ratio [OR]: 1.77, 95% confidence interval [CI]: 1.05-3.00, p= 0.034) while women carrying the TFPI -287C allele (TC + CC genotypes) had a protection effect on having RPL (OR: 0.46, 95% CI: 0.26-0.83, p= 0.009). The TCC haplotype for TFPI T-33C/ TFPI T-287C/ TFPI C-399T SNPs was less frequent in cases (5.7%) than in controls (11.6%) (OR: 0.45, 95% CI: 0.23-0.90, p= 0.025). In conclusion, our data indicate that SERPINC1 786G>A variant increases the risk for RPL, while TFPI T-287C variant is protective; however, further studies are required to confirm our findings.

  9. Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors

    SciTech Connect

    Hagiwara, Hiroki; Saito, Fumiaki; Masaki, Toshihiro; Ikeda, Miki; Nakamura-Ohkuma, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of TSA, one of most potent HDACIs, on myogenesis using the C2C12 skeletal muscle cell line. Black-Right-Pointing-Pointer TSA enhances the expression of myosin heavy chain without affecting DAPC expression. Black-Right-Pointing-Pointer TSA enhances the expression of the early MRFs, Myf5 and MEF2, and suppresses the late MRF, myogenin, after 24 h treatment. Black-Right-Pointing-Pointer TSA enhances the expression of the myogenic repressors, Ids, which inhibit myogenic differentiation. Black-Right-Pointing-Pointer TSA promotes myogenesis by coordinating the expression of MRFs and myogenic repressors. -- Abstract: Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.

  10. Complicated Whipple’s disease and endocarditis following tumor necrosis factor inhibitors

    PubMed Central

    Marth, Thomas

    2014-01-01

    AIM: To test whether treatment with tumor necrosis factor inhibitors (TNFI) is associated with complications of Tropheryma whipplei (T. whipplei) infection. METHODS: Because unexplained arthritis is often the first Whipple’s disease (WD) symptom, patients may undergo treatment with TNFI before diagnosis. This may influence the course of infection with T. whipplei, which causes WD, because host immune defects contribute to the pathogenesis of WD. A literature search and cross referencing identified 19 reports of TNFI treatment prior to WD diagnosis. This case-control study compared clinical data in patients receiving TNFI therapy (group I, n = 41) with patients not receiving TNFI therapy (group II, n = 61). Patients from large reviews served as controls (group III, n = 1059). RESULTS: The rate of endocarditis in patient group I was significantly higher than in patient group II (12.2% in group I vs 1.6% in group II, P < 0.05), and group III (12.2% in group I vs 0.16% in group III, P < 0.01). Other, severe systemic or local WD complications such as pericarditis, fever or specific organ manifestations were increased also in group I as compared to the other patient groups. However, diarrhea and weight loss were somewhat less frequent in patient group I. WD is typically diagnosed with duodenal biopsy and periodic acid Schiff (PAS) staining. PAS-stain as standard diagnostic test had a very high percentage of false negative results (diagnostic failure in 63.6% of cases) in group I. Polymerase chain reaction (PCR) for T. whipplei was more accurate than PAS-stainings (diagnostic accuracy, rate of true positive tests 90.9% for PCR vs 36.4% for PAS, P < 0.01). CONCLUSION: TNFI trigger severe WD complications, particularly endocarditis, and lead to false-negative PAS-tests. In case of TNFI treatment failure, infection with T. whipplei should be considered. PMID:25548618

  11. [Pyramiding of senescence-inhibition IPT gene and Xa23 for resistance to bacterial blight in rice (Oryza sativa L.)].

    PubMed

    He, Guang-Ming; Sun, Chuan-Qing; Fu, Yong-Cai; Fu, Qiang; Zhao, Kai-Jun; Wang, Chun-Lian; Zhang, Qi; Ling, Zhong-Zhuan; Wang, Xiang-Kun

    2004-08-01

    Transgenic lines (GC-1) carrying a senescence-inhibition cheimeric gene, IPT (isopentenyl transferase) gene, CBB23, a isogenic lines carrying Xa23 gene for resistance to bacterial blight, and Hexi15, a commercial cultivar showing high resistance to blast disease, were used as donors to pyramid IPT gene and Xa23 by marker-assisted selection (MAS). Seventeen BC1F1 plants pyramiding Xa23 gene and IPT genes were obtained from three multi-cross combinations. Then, the plants carrying Xa23 and IPT genes were crossed with parental lines of two-line hybrid rice, such as 9311, E32, Pei' ai 64S and W9834S. The progenies were backcrossed the acceptor parents. A total of 17 plants carrying Xa23 and IPT genes were detected by PCR, disease resistance identification and analysis of CTK contents of in the four combinations of "(9311///Hexi15/CBB23// GC-1) x 9311", "(E32///Hexi15/CBB23//GC-1) x E32", "(Pei'ai 64S///Hexi15/CBB23//GC-1) x Pei' ai 64S" and "(GC-1/CBB23//W9834S/Hexi15) x W9834S". These plants showed resistance to blast disease by inoculating test using 21 the lines of Pyricularia grisea from Northern China. Six plants of BC2F1 pyramiding Xa23 and IPT genes were further obtained in the combinations of "[(9311///Hexi15/CBB23//GC-1) x 9311] x 9311", "[(E32///Hexi15/CBB23//GC-1) x E32] x E32". After backcrossed and self-crossed 1 approximately 2nd, the plants pyramiding Xa23 and IPT genes can be used in the program of hybrid rice breeding.

  12. Novel chemo-enzymatic oligomers of cinnamic acids as direct and indirect inhibitors of coagulation proteinases.

    PubMed

    Monien, Bernhard H; Henry, Brian L; Raghuraman, Arjun; Hindle, Michael; Desai, Umesh R

    2006-12-01

    Thrombin and factor Xa, two important procoagulant enzymes, have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. Our efforts on exploiting the indirect mechanism led us to study a carboxylic acid-based scaffold, which displayed major acceleration in the inhibition of these enzymes [J. Med. Chem.2005, 48, 1269, 5360]. This work advances the study to chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids, DHPs, which display interesting anticoagulant properties. Oligomers, ranging in size from tetramers to pentadecamers, were prepared through peroxidase-catalyzed oxidative coupling of caffeic, ferulic, and sinapic acids, and sulfated using triethylamine-sulfur trioxide complex. Chromatographic, spectroscopic, and elemental studies suggest that the DHPs are heterogeneous, polydisperse preparations composed of inter-monomer linkages similar to those found in natural lignins. Measurement of activated thromboplastin and prothrombin time indicates that both the sulfated and unsulfated derivatives of the DHPs display anticoagulant activity, which is dramatically higher than that of the reference polyacrylic acids. More interestingly, this activity approaches that of low-molecular-weight heparin with the sulfated derivative showing approximately 2- to 3-fold greater potency than the unsulfated parent. Studies on the inhibition of factor Xa and thrombin indicate that the oligomers exert their anticoagulant effect through both direct and indirect inhibition mechanisms. This dual inhibition property of 4-hydroxycinnamic acid-based DHP oligomers is the first example in inhibitors of coagulation. This work puts forward a novel, non-heparin structure, which may be exploited for the design of potent, dual action inhibitors of coagulation through combinatorial virtual screening on a library of DHP oligomers.

  13. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.

  14. The development of novel inhibitors of tumor necrosis factor-alpha production based on substituted [5,5]-bicyclic pyrozolones

    SciTech Connect

    Laufersweiler, Matthew; Brugel, Todd; Clark, Michael; Golebiowski, Adam; Bookland, Roger; Laughlin, Steven; Sabat, Mark; Townes, Jennifer; VanRens, John; De, Biswanath; Hsieh, Lily; Heitmeyer, Sandra; Juergens, Karen; Brown, Kimberly; Mekel, Marlene; Walter, Richard; Janusz, Michael

    2010-11-16

    Novel substituted [5,5]-bicyclic pyrzazolones are presented as inhibitors of tumor necrosis factor-{alpha} (TNF-{alpha}) production. Many of these compounds show low nanomolar activity against lipopolysaccaride (LPS)-induced TNF-{alpha} production in THP-1 cells. This class of molecules was co-crystallized with mutated p38, and several analogs showed good oral bioavailability in the rat. Oral activity of these compounds in the rat iodoacetate model for osteoarthritis is discussed.

  15. Initial Assessment, Surveillance, and Management of Blood Pressure in Patients Receiving Vascular Endothelial Growth Factor Signaling Pathway Inhibitors

    PubMed Central

    Bakris, George L.; Black, Henry R.; Chen, Helen X.; Durand, Jean-Bernard; Elliott, William J.; Ivy, S. Percy; Leier, Carl V.; Lindenfeld, JoAnn; Liu, Glenn; Remick, Scot C.; Steingart, Richard; Tang, W. H. Wilson

    2010-01-01

    Hypertension is a mechanism-based toxic effect of drugs that inhibit the vascular endothelial growth factor signaling pathway (VSP). Substantial evidence exists for managing hypertension as a chronic condition, but there are few prospectively collected data on managing acute hypertension caused by VSP inhibitors. The Investigational Drug Steering Committee of the National Cancer Institute convened an interdisciplinary cardiovascular toxicities expert panel to evaluate this problem, to make recommendations to the Cancer Therapy Evaluation Program on further study, and to structure an approach for safe management by treating physicians. The panel reviewed: the published literature on blood pressure (BP), hypertension, and specific VSP inhibitors; abstracts from major meetings; shared experience with the development of VSP inhibitors; and established principles of hypertension care. The panel generated a consensus report including the recommendations on clinical concerns summarized here. To support the greatest possible number of patients to receive VSP inhibitors safely and effectively, the panel had four recommendations: 1) conduct and document a formal risk assessment for potential cardiovascular complications, 2) recognize that preexisting hypertension will be common in cancer patients and should be identified and addressed before initiation of VSP inhibitor therapy, 3) actively monitor BP throughout treatment with more frequent assessments during the first cycle of treatment, and 4) manage BP with a goal of less than 140/90 mmHg for most patients (and to lower, prespecified goals in patients with specific preexisting cardiovascular risk factors). Proper agent selection, dosing, and scheduling of follow-up should enable maintaining VSP inhibition while avoiding the complications associated with excessive or prolonged elevation in BP. PMID:20351338

  16. Guinea pig macroalbumin. A major inhibitor of activated Hageman factor in plasma with an alpha 2-macroglobulin-like nature.

    PubMed Central

    Ishimatsu, T.; Yamamoto, T.; Kozono, K.; Kambara, T.

    1984-01-01

    A major inhibitor of the beta form of activated Hageman factor (beta-HFa) with an apparent molecular weight of 28,000, which was reported as a strong permeability factor (Yamamoto and Cochrane, Am J Pathol 1981, 105: 164-175), was purified from guinea pig plasma. When it was depleted in vitro, the plasma lost 71% of the total inhibitory activity toward beta-HFa. The inhibitor, termed "macroalbumin," with an apparent molecular weight of 720,000 and an apparent pI of 4.6, seemed to be an inhibitor similar to alpha 1- or alpha 2-macroglobulin of man and other mammalian species in physicochemical characteristics and in enzymologic properties. Though the inhibitory activity to beta-HFa was negligible in normal skin extract, a significant inhibitory activity appeared in extracts of permeability-enhanced skin sites which were induced by intradermal beta-HFa injection. The inhibitory activity that appeared was macroalbumin-dependent, with more than a 10-fold increase in the concentration. These results indicate the roles of macroalbumin as a negative feedback regulator in situ to the Hageman-factor-dependent pathway in a permeability enhancement system. Images Figure 4 Figure 5 PMID:6201075

  17. Histone deacetylase inhibitors decrease Toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment

    PubMed Central

    Bode, Konrad A; Schroder, Kate; Hume, David A; Ravasi, Timothy; Heeg, Klaus; Sweet, Matthew J; Dalpke, Alexander H

    2007-01-01

    Post-translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibited induction of both interleukin-12 protein p40 (IL-12p40) mRNA and protein upon stimulation of Toll-like receptors (TLRs). Moreover, TLR-mediated up-regulation of costimulatory molecules was also inhibited. Up-regulation of tumour necrosis factor-α mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL-1β was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL-12p40 locus. Nevertheless, IL-12p40 chromatin remodelling, binding of Rel-A and IRF1 to the IL-12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor-κB and mitogen-activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment. PMID:17635610

  18. Explicit polynomial formulas for solutions of the matrix equation AX-XA=C

    SciTech Connect

    Demenchuk, Alexandr K.; Makarov, Evgenii K.

    2009-08-15

    We provide some mathematical tool to analyze the possible theoretical structure of Hamiltonian reconstructed from von Neumann or Heisenberg's equation for a finite-dimensional quantum system. To this end, we give an explicit polynomial representation for the general solution of the matrix equation AX-XA=C together with some (also polynomial) solvability conditions when A and C are some complex square matrices of the same size and the matrix A is semisimple. When the matrix A is normal, we derive a polynomial existence condition for Hermitian solutions and a similar formula for all solutions of this type.

  19. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  20. Gene silencing using the recessive rice bacterial blight resistance gene xa13 as a new paradigm in plant breeding.

    PubMed

    Li, Changyan; Wei, Jing; Lin, Yongjun; Chen, Hao

    2012-05-01

    Resistant germplasm resources are valuable for developing resistant varieties in agricultural production. However, recessive resistance genes are usually overlooked in hybrid breeding. Compared with dominant traits, however, they may confer resistance to different pathogenic races or pest biotypes with different mechanisms of action. The recessive rice bacterial blight resistance gene xa13, also involved in pollen development, has been cloned and its resistance mechanism has been recently characterized. This report describes the conversion of bacterial blight resistance mediated by the recessive xa13 gene into a dominant trait to facilitate its use in a breeding program. This was achieved by knockdown of the corresponding dominant allele Xa13 in transgenic rice using recently developed artificial microRNA technology. Tissue-specific promoters were used to exclude most of the expression of artificial microRNA in the anther to ensure that Xa13 functioned normally during pollen development. A battery of highly bacterial blight resistant transgenic plants with normal seed setting rates were acquired, indicating that highly specific gene silencing had been achieved. Our success with xa13 provides a paradigm that can be adapted to other recessive resistance genes.

  1. The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity

    PubMed Central

    Caddell, Daniel F.; Wei, Tong; Park, Chang-Jin; Ronald, Pamela C.

    2016-01-01

    Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression. PMID:27525297

  2. Factor VIII brand and the incidence of factor VIII inhibitors in previously untreated UK children with severe hemophilia A, 2000-2011.

    PubMed

    Collins, Peter W; Palmer, Benedict P; Chalmers, Elizabeth A; Hart, Daniel P; Liesner, Ri; Rangarajan, Savita; Talks, Katherine; Williams, Michael; Hay, Charles R M

    2014-11-27

    The effect of recombinant factor VIII (rFVIII) brand on inhibitor development was investigated in all 407 severe hemophilia A previously untreated patients born in the United Kingdom (UK) between 1 January 2000 and 31 December 2011. Eighty-eight (22%) had been in the RODIN study. Information was extracted from the National Haemophilia Database. Because exposure days (EDs) were not known for some patients, time from first treatment was used as a surrogate for rFVIII exposure. An inhibitor developed in 118 (29%) patients, 60 high and 58 low titer, after a median (interquartile range) of 7.8 (3.3-13.5) months from first exposure and 16 (9-30) EDs. Of 128 patients treated with Kogenate Bayer/Helixate NexGen, 45 (35.2%, 95% confidence interval [CI] 27.4-43.8) developed an inhibitor compared with 42/172 (24.4%, 95% CI 18.6% to 31.4%) with Advate (P = .04). The adjusted hazard ratio (HR) (95% CI) for Kogenate Bayer/Helixate NexGen compared with Advate was 2.14 (1.12-4.10) (P = .02) for high titer and 1.75 (1.11-2.76) (P = .02) for all inhibitors. When excluding UK-RODIN patients, the adjusted HR (95% CI) for high-titer inhibitors was 2.00 (0.93-4.34) (P = .08). ReFacto AF was associated with a higher incidence of all, but not high-titer, inhibitors than Advate. These results will help inform debate around the relative immunogenicity and use of rFVIII brands.

  3. Fibroblast Growth Factor (FGF) Receptor/FGF Inhibitors: Novel Targets and Strategies for Optimization of Response of Solid Tumors.

    PubMed

    Hierro, Cinta; Rodon, Jordi; Tabernero, Josep

    2015-12-01

    The fibroblast growth factor receptor (FGFR) pathway plays a major role in several biological processes, from organogenesis to metabolism homeostasis and angiogenesis. Several aberrations, including gene amplifications, point mutations, and chromosomal translocations have been described across solid tumors. Most of these molecular alterations promote multiple steps of carcinogenesis in FGFR oncogene-addicted cells, increasing cell proliferation, angiogenesis, and drug resistance. Data suggest that upregulation of FGFR signaling is a common event in many cancer types. The FGFR pathway thus arises as a potential promising target for cancer treatment. Several FGFR inhibitors are currently under development. Initial preclinical results have translated into limited successful clinical responses when first-generation, nonspecific FGFR inhibitors were evaluated in patients. The future development of selective and unselective FGFR inhibitors will rely on a better understanding of the tissue-specific role of FGFR signaling and identification of biomarkers to select those patients who will benefit the most from these drugs. Further studies are warranted to establish the predictive significance of the different FGFR-aberrations and to incorporate them into clinical algorithms, now that second-generation, selective FGFR inhibitors exist.

  4. Immune response to influenza vaccination in children treated with methotrexate or/and tumor necrosis factor-alpha inhibitors.

    PubMed

    Woerner, Andreas; Sauvain, Marie-Josèphe; Aebi, Christoph; Otth, Margrit; Bolt, Isabel B

    2011-12-01

    In children treated with immunosuppressive medication such as methotrexate and tumor necrosis factor-alpha (TNF-α) inhibitors, additional immunizations are recommended because of increased susceptibility to infections. However, it is unclear if adequate antibody response to vaccinations can be established in children receiving methotrexate and/or TNF-α inhibitors. In a prospective open label study, we assessed seroprotection and seroconversion following influenza vaccination during 2 seasons (6 strains) in 36 children with autoimmune disease treated either with methotrexate (n=18), TNF-α inhibitors (n=10) or both (n=8) and a control group of 16 immunocompetent children. Influenza antibody titers were determined by hemagglutinin inhibition assay, before and 4-8 weeks after vaccination. Post-vaccination seroprotection (defined as a titer ≥1:40) did not significantly differ between immunosuppressed and immunocompetent subjects. Seroconversion, defined as the change from a nonprotective (< 1:40) to a protective titer (≥1:40) with at least a 4-fold titer increase, was less likely to occur in immunosuppressed patients, although no significant difference from the control group was established. Safety evaluation of vaccination showed no serious adverse events. Children receiving methotrexate and/or TNF-α inhibitors can be safely and effectively immunized against influenza, with a seroprotection after vaccination comparable to immunocompetent children.

  5. SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

    PubMed

    Ferreira, Viviana P; Fazito Vale, Vladimir; Pangburn, Michael K; Abdeladhim, Maha; Mendes-Sousa, Antonio Ferreira; Coutinho-Abreu, Iliano V; Rasouli, Manoochehr; Brandt, Elizabeth A; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Pereira, Marcos Horácio; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M C; Gontijo, Nelder F; Collin, Nicolas; Valenzuela, Jesus G

    2016-01-13

    Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

  6. SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

    PubMed Central

    Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

    2016-01-01

    Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

  7. The prevalence of factor VIII and IX inhibitors among Saudi patients with hemophilia: Results from the Saudi national hemophilia screening program.

    PubMed

    Owaidah, Tarek; Momen, Abdulkareem Al; Alzahrani, Hazzaa; Almusa, Abdulrahman; Alkasim, Fawaz; Tarawah, Ahmed; Nouno, Randa Al; Batniji, Fatima Al; Alothman, Fahad; Alomari, Ali; Abu-Herbish, Saud; Abu-Riash, Mahmoud; Siddiqui, Khawar; Ahmed, Mansor; Mohamed, S Y; Saleh, Mahasen

    2017-01-01

    Hemophilia A and B are X-linked diseases that predominantly affect male patients. Patients can develop coagulation factor inhibitors, which exponentially increases the treatment cost. However, the prevalence of factor VIII and IX inhibitors in Saudi Arabia is unclear.This study aimed to determine the Saudi prevalence of factor VIII and IX inhibitors.This 4-year, 7-center, cross-sectional study evaluated the Saudi prevalences of hemophilia A and B. We collected the patients' clinical data, evaluated their disease, and tested for factor inhibitors.We included 202 patients with hemophilia (median age at diagnosis: 0.13 years, range: birth-34.8 years). The patients included 198 male patients (98%), 148 patients with hemophilia A (73.3%), and 54 patients with hemophilia B (26.7%). The patients exhibited severe factor VIII activity (<1%; 121 patients; 5.2%), moderate activity (1-5%; 7 patients; 4.9%), and mild activity (14 patients; 9.9%). Among the patients with care-related data, most patients were treated for episodic bleeding (76.8%) or received prophylaxis (22.6%); 1 patient received both treatments. Among the patients with source-related data, the factor replacements were derived from plasma (48.4%), recombinant concentrates (22.9%), both sources (14.6%), or fresh frozen plasma (14.1%). Factor VIII inhibitors were observed in 43 (29.3%) of the 147 patients, and only 1 of the 54 patients developed factor IX inhibitors. Most patients who developed inhibitors had severe hemophilia (40/44; 90.9%), and inhibitors were also common among patients who received recombinant products (14/43; 32.6%).The Saudi prevalence of factor inhibitors was similar to those among other ethnic populations.

  8. Association Between Ischemic Stroke and Tumor Necrosis Factor Inhibitor Therapy in Patients With Rheumatoid Arthritis

    PubMed Central

    Low, Audrey S. L.; Lunt, Mark; Mercer, Louise K.; Watson, Kath D.; Dixon, William G.; Symmons, Deborah P. M.

    2016-01-01

    Objective Patients with rheumatoid arthritis (RA) are at an increased risk of ischemic stroke. Tumor necrosis factor inhibitors (TNFi) may influence risk and mortality after ischemic stroke by reducing inflammation. This study was undertaken to examine the association of TNFi with the risk of incident ischemic stroke and with 30‐day and 1‐year mortality after ischemic stroke. Methods Patients with RA starting therapy with TNFi and a biologics‐naive comparator group treated with synthetic disease‐modifying antirheumatic drugs (DMARDs) only were recruited to the British Society for Rheumatology Biologics Register for Rheumatoid Arthritis from 2001 to 2009. Patients were followed up via clinical and patient questionnaires as well as the national death register. Incident strokes were classified as ischemic if brain imaging reports suggested ischemia or if ischemic stroke was reported as the underlying cause of death on a death certificate. Patients with a previous stroke were excluded. Risk of ischemic stroke was compared between patients receiving synthetic DMARDs only and those ever‐exposed to TNFi using a Cox proportional hazards regression model adjusted for potential confounders. Mortality after ischemic stroke was compared between synthetic DMARD–treated patients and TNFi‐treated patients using logistic regression, adjusted for age and sex. Results To April 2010, 127 verified incident ischemic strokes (21 in 3,271 synthetic DMARD–treated patients and 106 in 11,642 TNFi‐treated patients) occurred during 11,973 and 61,226 person‐years of observation, respectively (incidence rate 175 versus 173 per 100,000 person‐years). After adjustment for confounders, there was no association between ever‐exposure to TNFi and ischemic stroke (hazard ratio 0.99 [95% confidence interval (95% CI) 0.54–1.81]). Mortality 30 days or 1 year after ischemic stroke was not associated with concurrent TNFi exposure (odds ratio 0.18 [95% CI 0.03–1.21] and 0.60 [95

  9. The discovery of novel vascular endothelial growth factor receptor tyrosine kinases inhibitors: pharmacophore modeling, virtual screening and docking studies.

    PubMed

    Yu, Hui; Wang, Zhanli; Zhang, Liangren; Zhang, Jufeng; Huang, Qian

    2007-03-01

    We have applied pharmacophore generation, database searching and docking methodologies to discover new structures for the design of vascular endothelial growth factor receptors, the tyrosine kinase insert domain-containing receptor kinase inhibitors. The chemical function based pharmacophore models were built for kinase insert domain-containing receptor kinase inhibitors from a set of 10 known inhibitors using the algorithm HipHop, which is implemented in the CATALYST software. The highest scoring HipHop model consists of four features: one hydrophobic, one hydrogen bond acceptor, one hydrogen bond donor and one ring aromatic function. Using the algorithm CatShape within CATALYST, the bound conformation of 4-amino-furo [2, 3-d] pyrimidine binding to kinase insert domain-containing receptor kinase was used to generate a shape query. A merged shape and hypothesis query that is in an appropriate alignment was then built. The combined shape and hypothesis model was used as a query to search Maybridge database for other potential lead compounds. A total of 39 compounds were retrieved as hits. The hits obtained were docked into kinase insert domain-containing receptor kinase active site. One novel potential lead was proposed based on CATALYST fit value, LigandFit docking scores, and examination of how the hit retain key interactions known to be required for kinase binding. This compound inhibited vascular endothelial growth factor stimulated kinase insert domain-containing receptor phosphorylation in human umbilical vein endothelial cells.

  10. Beyond stopping the bleed: short-term episodic prophylaxis with recombinant activated factor FVII in haemophilia patients with inhibitors

    PubMed Central

    Šalek, Silva Zupančić; Auerswald, Günter; Benson, Gary; Dolan, Gerry; Duffy, Anne; Hermans, Cedric; Jiménez-Yuste, Victor; Ljung, Rolf; Morfini, Massimo; Santagostino, Elena; Lambert, Thierry

    2017-01-01

    Preventing haemarthroses and arthropathy is a major challenge in patients with haemophilia and inhibitors, as treatment options are limited. One potential strategy is short-term episodic prophylaxis, which extends bypassing agent therapy beyond the resolution of bleeding to include the post-bleed inflammatory phase. At the 13th Zürich Haemophilia Forum, an expert panel reviewed the rationale behind this strategy, explored its current use with recombinant activated factor VII (rFVIIa) and considered treatment monitoring and optimisation. Two protocols are currently used for short-term episodic prophylaxis, both of which stipulate on-demand rFVIIa until resolution of bleeding, followed by daily dosing for ≥3 days to prevent re-bleeds. Short-term episodic prophylaxis should be individualised to optimise outcomes, perhaps through early treatment initiation or by combining rFVIIa with other treatments (e.g. factor VIII, tranexamic acid). Encouraging treatment compliance can also improve outcomes. Additionally, there is a need to develop objective clinical outcome measures, biomarkers and imaging protocols that can monitor treatment outcomes and joint disease in patients with inhibitors. A proactive approach incorporating a systematic package of care is needed. Currently, short-term episodic prophylaxis with rFVIIa may be an alternative treatment option to on-demand treatment for patients with inhibitors. PMID:26674816

  11. Exploitation of Castration-Resistant Prostate Cancer Transcription Factor Dependencies by the Novel BET Inhibitor ABBV-075.

    PubMed

    Faivre, Emily J; Wilcox, Denise; Lin, Xiaoyu; Hessler, Paul; Torrent, Maricel; He, Wei; Uziel, Tamar; Albert, Daniel H; McDaniel, Keith; Kati, Warren; Shen, Yu

    2017-01-01

    Competitive inhibitors of acetyl-lysine binding to the bromodomains of the BET (bromodomain and extra terminal) family are being developed for the treatment of solid and hematologic malignancies. The function of BET family member BRD4 at enhancers/superenhancers has been shown to sustain signal-dependent or pathogenic gene expression programs. Here, the hypothesis was tested that the transcription factor drivers of castration-resistant prostate cancer (CRPC) clinical progression, including the androgen receptor (AR), are critically dependent on BRD4 and thus represent a sensitive solid tumor indication for the BET inhibitor ABBV-075. DHT-stimulated transcription of AR target genes was inhibited by ABBV-075 without significant effect on AR protein expression. Furthermore, ABBV-075 disrupted DHT-stimulated recruitment of BET family member BRD4 to gene-regulatory regions cooccupied by AR, including the well-established PSA and TMPRSS2 enhancers. Persistent BET inhibition disrupted the composition and function of AR-occupied enhancers as measured by a reduction in AR and H3K27Ac ChIP signal and inhibition of enhancer RNA transcription. ABBV-075 displayed potent antiproliferative activity in multiple models of resistance to second-generation antiandrogens and inhibited the activity of the AR splice variant AR-V7 and ligand-binding domain gain-of-function mutations, F877L and L702H. ABBV-075 was also a potent inhibitor of MYC and the TMPRSS2-ETS fusion protein, important parallel transcription factor drivers of CRPC.

  12. Metabolic factors, adipose tissue, and plasminogen activator inhibitor-1 levels in Type 2 diabetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasminogen activator inhibitor-1 (PAI-1) production by adipose tissue is increased in obesity, and its circulating levels are high in type 2 diabetes. PAI-1 increases cardiovascular risk by favoring clot stability, interfering with vascular remodeling, or both. We investigated in obese diabetic per...

  13. Tissue factor pathway inhibitor for prediction of placenta-mediated adverse pregnancy outcomes in high-risk women: AngioPred study

    PubMed Central

    Di Bartolomeo, Aurélie; Chauleur, Céline; Gris, Jean-Christophe; Chapelle, Céline; Noblot, Edouard; Laporte, Silvy

    2017-01-01

    Objective The study aimed to evaluate if the rate of tissue factor pathway inhibitor during pregnancy and following delivery could be a predictive factor for placenta-mediated adverse pregnancy outcomes in high-risk women. Methods This was a prospective multicentre cohort study of 200 patients at a high risk of occurrence or recurrence of placenta-mediated adverse pregnancy outcomes conducted between June 2008 and October 2010. Measurements of tissue factor pathway inhibitor resistance (normalized ratio) and tissue factor pathway inhibitor activity were performed for the last 72 patients at 20, 24, 28, 32, and 36 weeks of gestation and during the postpartum period. Results Overall, 15 patients presented a placenta-mediated adverse pregnancy outcome. There was no difference in normalized tissue factor pathway inhibitor ratios between patients with and without placenta-mediated adverse pregnancy outcomes during pregnancy and in the post-partum period. Patients with placenta-mediated adverse pregnancy outcomes had tissue factor pathway inhibitor activity rates that were significantly higher than those in patients without at as early as 24 weeks of gestation. The same results were observed following delivery. Conclusion Among high-risk women, the tissue factor pathway inhibitor activity of patients with gestational vascular complications is higher than that in other patients. Hence, these markers could augment a screening strategy that includes an analysis of angiogenic factors as well as clinical and ultrasound imaging with Doppler measurement of the uterine arteries. PMID:28328938

  14. Concurrent Intervention With Exercises and Stabilized Tumor Necrosis Factor Inhibitor Therapy Reduced the Disease Activity in Patients With Ankylosing Spondylitis

    PubMed Central

    Liang, Hui; Li, Wen-Rong; Zhang, Hua; Tian, Xu; Wei, Wei; Wang, Chun-Mei

    2015-01-01

    Abstract Since the use of tumor necrosis factor (TNF) inhibitor therapy is becoming wider, the effects of concurrent intervention with exercises and stabilized TNF inhibitors therapy in patients with ankylosing spondylitis (AS) are different. The study aimed to objectively evaluate whether concurrent intervention with exercises and stabilized TNF inhibitors can reduce the disease activity in patients with AS. A search from PubMed, Web of Science, EMBASE, and the Cochrane Library was electronically performed to collect studies which compared concurrent intervention with exercise and TNF inhibitor to conventional approach in terms of disease activity in patients with AS published from their inception to June 2015. Studies that measured the Bath Ankylosing Spondylitis Functional Index (BASFI), the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Bath Ankylosing Spondylitis Metrology Index (BASMI), and chest expansion as outcomes were included. Two independent investigators screened the identified articles, extracted the data, and assessed the methodological quality of the included studies. Quantitative analysis was performed with Review Manager (RevMan) software (version 5.3.0). A total of 5 studies comprising 221 participants were included in the study. Meta-analyses showed that concurrent intervention with exercises and stabilized TNF inhibitors therapy significantly reduced the BASMI scores (MD, −0.99; 95% CI, −1.61 to −0.38) and BASDAI scores (MD, −0.58; 95% CI, −1.10 to −0.06), but the BASFI scores (MD, −0.31; 95% CI, −0.76 to 0.15) was not reduced, and chest expansion (MD, 0.80; 95% CI, −0.18 to 1.78) was not increased. Concurrent intervention with exercises and stabilized TNF inhibitors therapy can reduce the disease activity in patients with AS. More randomized controlled trials (RCTs) with high-quality, large-scale, and appropriate follow-up are warranted to further establish the benefit of concurrent intervention with

  15. Prediction of Inhibitory Activity of Epidermal Growth Factor Receptor Inhibitors Using Grid Search-Projection Pursuit Regression Method

    PubMed Central

    Du, Hongying; Hu, Zhide; Bazzoli, Andrea; Zhang, Yang

    2011-01-01

    The epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK) is an important protein target for anti-tumor drug discovery. To identify potential EGFR inhibitors, we conducted a quantitative structure–activity relationship (QSAR) study on the inhibitory activity of a series of quinazoline derivatives against EGFR tyrosine kinase. Two 2D-QSAR models were developed based on the best multi-linear regression (BMLR) and grid-search assisted projection pursuit regression (GS-PPR) methods. The results demonstrate that the inhibitory activity of quinazoline derivatives is strongly correlated with their polarizability, activation energy, mass distribution, connectivity, and branching information. Although the present investigation focused on EGFR, the approach provides a general avenue in the structure-based drug development of different protein receptor inhibitors. PMID:21811593

  16. Recreational Use of Phosphodiesterase 5 Inhibitors and Its Associated Factors among Undergraduate Male Students in an Ethiopian University: A Cross-Sectional Study

    PubMed Central

    Bhagavathula, Akshaya Srikanth; Gebresillassie, Begashaw Melaku; Tefera, Yonas Getaye; Belachew, Sewunet Admasu; Erku, Daniel Asfaw

    2016-01-01

    Purpose To assess the prevalence of phosphodiesterase 5 (PDE5) inhibitor use and associated factors among University of Gondar undergraduate students. Materials and Methods An institution-based, cross-sectional study, using a survey questionnaire, was conducted from October to December 2015 to assess PDE5 inhibitor use and associated factors among male students at the University of Gondar. A Self-Esteem and Relationship questionnaire (14 items), an International Index of Erectile Function questionnaire (15 items) and a questionnaire on PDE5 inhibitor use (14 items) were included in the survey. Results Across all respondents (age, 21.9±1.88 years), more than half (55.7%, n=233) had heard about PDE5 inhibitors, but only 23 men (5.5%) reported trying a PDE5 inhibitor drug at least once. Older students were more likely to use PDE5 inhibitors compared to younger students (adjusted odds ratio [AOR], 1.40; 95% confidence interval [CI], 1.109~1.768). Those students who were smokers were 5.15 times more likely to use PDE5 inhibitors as compared to their non-smoking counterparts (AOR, 5.15; 95% CI, 2.096~12.687). In addition, multivariate logistic regression showed that being in a relationship, alcohol use, greater number of cigarettes smoked per day, and more sexual partners were significantly associated with PDE5 inhibitor use. Conclusions The prevalence of PDE5 inhibitor use among undergraduate students was 5.5%. Cigarette smoking and other substance use, older age, and greater number of sexual partners were significantly associated factors for PDE5 inhibitor use. These findings suggest that restricting access to PDE5 inhibitor drugs is essential to curtailing misuse among university students. PMID:28053948

  17. Predictive factors of response to mTOR inhibitors in neuroendocrine tumours.

    PubMed

    Zatelli, Maria Chiara; Fanciulli, Giuseppe; Malandrino, Pasqualino; Ramundo, Valeria; Faggiano, Antongiulio; Colao, Annamaria

    2016-03-01

    Medical treatment of neuroendocrine tumours (NETs) has drawn a lot of attention due to the recent demonstration of efficacy of several drugs on progression-free survival, including somatostatin analogs, small tyrosine kinase inhibitors and mTOR inhibitors (or rapalogs). The latter are approved as therapeutic agents in advanced pancreatic NETs and have been demonstrated to be effective in different types of NETs, with variable efficacy due to the development of resistance to treatment. Early detection of patients that may benefit from rapalogs treatment is of paramount importance in order to select the better treatment and avoid ineffective and expensive treatments. Predictive markers for therapeutic response are under intensive investigation, aiming at a tailored patient management and more appropriate resource utilization. This review summarizes the available data on the tissue, circulating and imaging markers that are potentially predictive of rapalog efficacy in NETs.

  18. HDAC and Proteasome Inhibitors Synergize to Activate Pro-Apoptotic Factors in Synovial Sarcoma

    PubMed Central

    Barrott, Jared J.; Yao, Ren Jie; Poulin, Neal M.; Brodin, Bertha A.; Jones, Kevin B.; Underhill, T. Michael; Nielsen, Torsten O.

    2017-01-01

    Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting its driving SS18-SSX fusion oncoprotein are currently available. Patients remain at high risk for early and late metastasis. A high-throughput drug screen consisting of over 900 tool compounds and epigenetic modifiers, representing over 100 drug classes, was undertaken in a panel of synovial sarcoma cell lines to uncover novel sensitizing agents and targetable pathways. Top scoring drug categories were found to be HDAC inhibitors and proteasomal targeting agents. We find that the HDAC inhibitor quisinostat disrupts the SS18-SSX driving protein complex, thereby reestablishing expression of EGR1 and CDKN2A tumor suppressors. In combination with proteasome inhibition, HDAC inhibitors synergize to decrease cell viability and elicit apoptosis. Quisinostat inhibits aggresome formation in response to proteasome inhibition, and combination treatment leads to elevated endoplasmic reticulum stress, activation of pro-apoptotic effector proteins BIM and BIK, phosphorylation of BCL-2, increased levels of reactive oxygen species, and suppression of tumor growth in a murine model of synovial sarcoma. This study identifies and provides mechanistic support for a particular susceptibility of synovial sarcoma to the combination of quisinostat and proteasome inhibition. PMID:28056055

  19. Bleeding in patients using new anticoagulants or antiplatelet agents: risk factors and management.

    PubMed

    Levi, M M; Eerenberg, E; Löwenberg, E; Kamphuisen, P W

    2010-02-01

    The most important adverse effect of antithrombotic treatment is the occurrence of bleeding. In case of serious or even life-threatening bleeding in a patient who uses anticoagulant agents or when patient on anticoagulants needs to undergo an urgent invasive procedure, anticoagulant treatment can be reversed by various specific strategies. Heparin and heparin derivatives can be counteracted by protamine sulphate, whereas the anticoagulant effect of vitamin K antagonists may be neutralised by administration of vitamin K or prothrombin complex concentrates. The antihaemostatic effect of aspirin and other antiplatelet strategies can be corrected by the administration of platelet concentrate and/or desmopressin, if needed. Recently, a new generation of anticoagulants with a greater specificity towards activated coagulation factors has been introduced and most of these agents are currently being evaluated in clinical studies, showing promising results. The new-generation anticoagulants include specific inhibitors of factor IIa or factor Xa (including pentasaccharides) and antiplatelet agents belonging to the class of thienopyridine derivatives. A limitation of the new class of anti-IIa and anti-Xa agents may be the lack of an appropriate strategy to reverse the effect if a bleeding event occurs, although in some cases the administration of recombinant factor VIIa may be an option.

  20. Identification of Iguratimod as an Inhibitor of Macrophage Migration Inhibitory Factor (MIF) with Steroid-sparing Potential.

    PubMed

    Bloom, Joshua; Metz, Christine; Nalawade, Saisha; Casabar, Julian; Cheng, Kai Fan; He, Mingzhu; Sherry, Barbara; Coleman, Thomas; Forsthuber, Thomas; Al-Abed, Yousef

    2016-12-16

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in a broad range of inflammatory and oncologic diseases. MIF is unique among cytokines in terms of its release profile and inflammatory role, notably as an endogenous counter-regulator of the anti-inflammatory effects of glucocorticoids. In addition, it exhibits a catalytic tautomerase activity amenable to the design of high affinity small molecule inhibitors. Although several classes of these compounds have been identified, biologic characterization of these molecules remains a topic of active investigation. In this study, we used in vitro LPS-driven assays to characterize representative molecules from several classes of MIF inhibitors. We determined that MIF inhibitors exhibit distinct profiles of anti-inflammatory activity, especially with regard to TNFα. We further investigated a molecule with relatively low anti-inflammatory activity, compound T-614 (also known as the anti-rheumatic drug iguratimod), and found that, in addition to exhibiting selective MIF inhibition in vitro and in vivo, iguratimod also has additive effects with glucocorticoids. Furthermore, we found that iguratimod synergizes with glucocorticoids in attenuating experimental autoimmune encephalitis, a model of multiple sclerosis. Our work identifies iguratimod as a valuable new candidate for drug repurposing to MIF-relevant diseases, including multiple sclerosis.

  1. Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib).

    PubMed

    Elkind, N Barry; Szentpétery, Zsófia; Apáti, Agota; Ozvegy-Laczka, Csilla; Várady, György; Ujhelly, Olga; Szabó, Katalin; Homolya, László; Váradi, András; Buday, László; Kéri, György; Német, Katalin; Sarkadi, Balázs

    2005-03-01

    Iressa (ZD1839, Gefitinib), used in clinics to treat non-small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signaling-dependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa.

  2. Isolation of hypoxia-inducible factor 1 (HIF-1) inhibitors from frankincense using a molecularly imprinted polymer.

    PubMed

    Lakka, Achillia; Mylonis, Ilias; Bonanou, Sophia; Simos, George; Tsakalof, Andreas

    2011-10-01

    Hypoxia-Inducible Factor 1 (HIF-1), a transcriptional activator, is highly involved in the pathology of cancer. Inhibition of HIF-1 retards tumor growth and enhances treatment efficiency when used in combination with chemo- or radiation therapy. The recent validation of HIF-1 as an important drug target in cancer treatment has stimulated efforts to identify and isolate natural or synthetic HIF-1 inhibitors. In the present study, quercetin, a known inhibitor of HIF-1, was imprinted in a polymer matrix in order to prepare a Molecularly Imprinted Polymer (MIP), which was subsequently used for the selective isolation of new inhibitors from frankincense, a gum resin used as anticancer remedy in traditional medicine. The frankincense components isolated by Solid Phase Extraction on MIP (MIP-SPE), efficiently inhibited the transcriptional activity of HIF-1 and decreased the protein levels of HIF-1α, the regulated subunit of HIF-1. The selective retention of acetyl 11-ketoboswellic acid (AKBA, one of the main bioactive components of frankincense) by MIP led to the revealing of its inhibitory activity on the HIF-1 signaling pathway. AKBA was selectively retained by SPE on the quercetin imprinted polymer, with an imprinting effect of 8.1 ± 4.6. Overall, this study demonstrates the potential of MIP application in the screening, recognition and isolation of new bioactive compounds that aim selected molecular targets, a potential that has been poorly appreciated until.

  3. MET inhibitor PHA-665752 suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells

    SciTech Connect

    Liu, Tongxin; Li, Qi; Sun, Quanquan; Zhang, Yuqin; Yang, Hua; Wang, Rong; Chen, Longhua; Wang, Wei

    2014-06-20

    Highlights: • We demonstrated that irradiation induced MET overexpression and activation. • The aberrant MET signal mediated by HGF induced proliferation and radioresistance of NPC cells. • MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. • PHA-665752 suppressed the three downstream pathway of HGF/MET signal in a dose-dependent manner. - Abstract: Although ionizing radiation (IR) has provided considerable improvements in nasopharyngeal carcinoma (NPC), in subsets of patients, radioresistance is still a major problem in the treatment. In this study, we demonstrated that irradiation induced MET overexpression and activation, and the aberrant MET signal mediated by hepatocyte growth factor (HGF) induced radioresistance. We also found that MET inhibitor PHA-665752 effectively suppressed HGF induced cell proliferation and radioresistance in NPC cells. Further investigation indicated that PHA-665752 suppressed the phosphorylation of the Akt, ERK1/2, and STAT3 proteins in a dose-dependent manner. Our data indicated that the combination of IR with a MET inhibitor, such as PHA-665752, might be a promising therapeutic strategy for NPC.

  4. Structure-Based Library Design and Fragment Screening for the Identification of Reversible Complement Factor D Protease Inhibitors.

    PubMed

    Vulpetti, Anna; Randl, Stefan; Rüdisser, Simon; Ostermann, Nils; Erbel, Paul; Mac Sweeney, Aengus; Zoller, Thomas; Salem, Bahaa; Gerhartz, Bernd; Cumin, Frederic; Hommel, Ulrich; Dalvit, Claudio; Lorthiois, Edwige; Maibaum, Jürgen

    2017-03-09

    Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.

  5. Risk factors for high-titer inhibitor development in children with hemophilia A: results of a cohort study.

    PubMed

    Halimeh, Susan; Bidlingmaier, Christoph; Heller, Christine; Gutsche, Sven; Holzhauer, Susanne; Kenet, Gili; Kurnik, Karin; Manner, Daniela; Iorio, Alfonso; Nowak-Göttl, Ulrike

    2013-01-01

    Among the discussed risk factors for high-titre inhibitor (HRI) development in patients with hemophilia A (HA) are high dose FVIII replacement therapy and use of recombinant FVIII concentrates (rFVIII). The aim of this study was to evaluate the aforementioned risk factors for HRI development in children with hemophilia A ≤2%. About 288 ascertained PUPs (Israel and Germany) were followed after initial HA diagnosis over 200 exposure days. Inhibitor-free survival, hazard ratios (HR), and 95% confidence intervals (CIs) were calculated. Adjustment was performed for factor VIII concentrates, median single dose over the first three months of treatment, first FVIII administration before the age of three months, presence of risk HA gene mutations, "intensive treatment moments" and "year of birth" (proxy for different treatment periods). HRI occurred in 71/288 children (24.7%). In multivariate analysis adjusted for "year of birth", underlying risk gene mutations (HR/CI: 2.37/1.40-3.99), FVIII dose, measured per one IU increase per kgbw (HR/CI: 1.05/1.04-1.07), and first FVIII administration before the age of three months showed a significant impact on HR development. The risk of HRI development was similar for recombinant or plasmatic FVIII products. Children at risk should be treated with carefully calculated lower dose regimens, adapted to individual bleeding situations.

  6. Effect of a 5-lipoxygenase inhibitor on nerve growth factor-induced thermal hyperalgesia in the rat.

    PubMed

    Amann, R; Schuligoi, R; Lanz, I; Peskar, B A

    1996-06-13

    Intraplantar injection of mouse beta (2.5S) nerve growth factor (NGF) caused thermal hyperalgesia and stimulated release of immunoreactive leukotriene B4 from the rat paw skin. Both effects of NGF were prevented by the 5-lipoxygenase inhibitor, (R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid (BAY X1005). BAY X1005 did not affect bradykinin-induced thermal hyperalgesia. These results suggest the participation of 5-lipoxygenase products of arachidonate in NGF-induced local thermal hyperalgesia.

  7. Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

    PubMed

    Vézina, C; Carcao, M; Infante-Rivard, C; Lillicrap, D; Stain, A M; Paradis, E; Teitel, J; Rivard, G E

    2014-11-01

    The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg(-1)  day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.

  8. Sensitivities to various epidermal growth factor receptor-tyrosine kinase inhibitors of uncommon epidermal growth factor receptor mutations L861Q and S768I: What is the optimal epidermal growth factor receptor-tyrosine kinase inhibitor?

    PubMed

    Banno, Eri; Togashi, Yosuke; Nakamura, Yu; Chiba, Masato; Kobayashi, Yoshihisa; Hayashi, Hidetoshi; Terashima, Masato; de Velasco, Marco A; Sakai, Kazuko; Fujita, Yoshihiko; Mitsudomi, Tetsuya; Nishio, Kazuto

    2016-08-01

    Most patients with non-small cell lung cancer (NSCLC) harboring common epidermal growth factor receptor (EGFR) mutations, such as deletions in exon 19 or the L858R mutation in exon 21, respond dramatically to EGFR tyrosine kinase inhibitors (EGFR-TKI), and their sensitivities to various EGFR-TKI have been well characterized. Our previous article showed the in vitro sensitivities of EGFR exon 18 mutations to EGFR-TKI, but little information regarding the sensitivities of other uncommon EGFR mutations is available. First, stable transfectant Ba/F3 cell lines harboring EGFR L858R (Ba/F3-L858R), L861Q (Ba/F3-L861Q) or S768I (Ba/F3-S768I) mutations were created and their drug sensitivities to various EGFR-TKI were examined. Both the Ba/F3-L861Q and Ba/F3-S768I cell lines were less sensitive to erlotinib, compared with the Ba/F3-L858R cell line, but their sensitivities to afatinib were similar to that of the Ba/F3-L858R cell line. The Ba/F3-L861Q cell line was similarly sensitive and the Ba/F3-S768I cell line was less sensitive to osimertinib, compared with the Ba/F3-L858R cell line. The results of western blot analyses were consistent with these sensitivities. Next, similar experiments were also performed using the KYSE270 (L861Q) and KYSE 450 (S768I) cell lines, and their results were compatible with those of the transfectant Ba/F3 cell lines. Our findings suggest that NSCLC harboring the EGFR L861Q mutation might be sensitive to afatinib or osimertinib and that NSCLC harboring the EGFR S768I mutation might be sensitive to afatinib. Overall, afatinib might be the optimal EGFR-TKI against these uncommon EGFR mutations.

  9. Early eradication of factor VIII inhibitor in patients with congenital hemophilia A by immune tolerance induction with a high dose of immunoglobulin.

    PubMed

    Mizoguchi, Yoko; Furue, Aya; Kagawa, Reiko; Chijimatsu, Ikue; Tomioka, Keita; Shimomura, Maiko; Imanaka, Yusuke; Nishimura, Shiho; Saito, Satoshi; Miki, Mizuka; Ono, Atsushi; Konishi, Nakao; Kawaguchi, Hiroshi; Kobayashi, Masao

    2016-04-01

    The production of factor VIII (FVIII) inhibitory antibodies is a serious problem in patients with hemophilia A. Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors and has been shown to be successful in 70 % of patients with hemophilia A. However, a minority of hemophilia patients present life-long inhibitors. To eliminate such inhibitors, we designed an intravenous immunoglobulin (IVIG) strategy in combination with high dose recombinant FVIII for ITI in hemophilia A children with inhibitors. Four previously untreated patients produced inhibitors within 16 exposures to FVIII. The peak inhibitor titers in these patients ranged from 3 to 14 BU/mL. The patients received ITI combined with IVIG within 1.5 months after the inhibitors were detected. All patients showed a negative titer for inhibitors by 28 days, with no anamnestic responses. The recovery of FVIII in the plasma concentration was normalized within three months after initiation of ITI. An additional course of IVIG administration led to induction of complete tolerance by 20 months after initiation of ITI therapy in all patients. ITI treatment with high-dose FVIII combined with IVIG may be effective for the early elimination of inhibitors.

  10. Discovery of Indenopyrazoles as a New Class of Hypoxia Inducible Factor (HIF)-1 Inhibitors

    PubMed Central

    2013-01-01

    The indenopyrazole framework was investigated as a new class of HIF-1α inhibitors. Indenopyrazole 2l was found to most strongly inhibit the hypoxia-induced HIF-1α transcriptional activity (IC50 = 0.014 μM) among all of the known compounds having relatively simple structures, unlike manassantins. Indenopyrazole 2l suppressed HIF-1α transcriptional activity without affecting both HIF-1α protein accumulation and HIF-1α/HIF-1β heterodimerization in nuclei under the hypoxic conditions, suggesting that 2l probably affected the transcriptional pathway induced by the HIF-1α/HIF-1β heterodimer. PMID:24900662

  11. Non-length-dependent and length-dependent small-fiber neuropathies associated with tumor necrosis factor (TNF)-inhibitor therapy in patients with rheumatoid arthritis: Expanding the spectrum of neurological disease associated with TNF-inhibitors

    PubMed Central

    Birnbaum, Julius; Bingham, Clifton O.

    2015-01-01

    Objective Small-fiber neuropathy causes severe burning pain, requires diagnostic approaches such as skin biopsy, and encompasses two subtypes based on distribution of neuropathic pain. Such biopsy-proven subtypes of small-fiber neuropathies have not been previously described as complications of tumor necrosis factor (TNF)-inhibitor therapy. Methods We therefore characterized clinical and skin biopsy findings in three rheumatoid arthritis (RA) patients who developed small-fiber neuropathies associated with TNF-inhibitors. We also conducted a systematic review of the literature to characterize subtypes of neuropathies previously reported in association with TNF-inhibitor therapy. Results Two patients presented with a “non-length-dependent” small-fiber neuropathy, experiencing unorthodox patterns of burning pain affecting the face, torso, and proximal extremities. Abnormal skin biopsy findings were limited to the proximal thigh, which is a marker of proximal-most dorsal root ganglia degeneration. In contrast, one patient presented with a “length-dependent” small-fiber neuropathy, experiencing burning pain only in the feet. Abnormal skin biopsy findings were limited to the distal feet, which is a marker of distal-most axonal degeneration. One patient developed a small-fiber neuropathy in the context of TNF-inhibitor-induced lupus. In all patients, neuropathies occurred during TNF-inhibitor-induced remission of RA disease activity and improved on withdrawal of TNF-inhibitors. Conclusions We describe a spectrum of small-fiber neuropathies not previously reported in association with TNF-inhibitor therapy, with clinical and skin biopsy findings suggestive of dorsal root ganglia as well as axonal degeneration. The development of small-fiber neuropathies during inactive joint disease and improvement of neuropathic pain upon withdrawal of TNF-inhibitor suggest a causative role of TNF-inhibitors. PMID:24439654

  12. Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type β and epidermal growth factor

    SciTech Connect

    Song, Xiaoling; Thalacker, F.W.; Nilsen-Hamilton, Marit

    2012-04-06

    The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type β (TGFβ) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGFβ regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGFβ also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGFβ pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGFβ are able to promote large and rapid changes in PAI-1 expression.

  13. XaNSoNS: GPU-accelerated simulator of diffraction patterns of nanoparticles

    NASA Astrophysics Data System (ADS)

    Neverov, V. S.

    XaNSoNS is an open source software with GPU support, which simulates X-ray and neutron 1D (or 2D) diffraction patterns and pair-distribution functions (PDF) for amorphous or crystalline nanoparticles (up to ∼107 atoms) of heterogeneous structural content. Among the multiple parameters of the structure the user may specify atomic displacements, site occupancies, molecular displacements and molecular rotations. The software uses general equations nonspecific to crystalline structures to calculate the scattering intensity. It supports four major standards of parallel computing: MPI, OpenMP, Nvidia CUDA and OpenCL, enabling it to run on various architectures, from CPU-based HPCs to consumer-level GPUs.

  14. Continuous infusion of porcine factor VIII in patients with haemophilia A and high-responding inhibitors: stability and clinical experience.

    PubMed

    O'Gorman, P; Dimichele, D M; Kasper, C K; Mannucci, P M; Santagostini, E; Hay, C R

    2001-11-01

    A multicentre retrospective survey was conducted to assess the efficacy and side-effect profile of porcine factor VIII (pFVIII:C) given by continuous infusion (CI) to patients with congenital haemophilia A and inhibitors. Twenty-nine episodes in 18 patients were treated by CI of pFVIII:C. Efficacy was graded as good in 79% of infusions and fair in 17%. There was a failed response in only one episode. Fourteen percent of patients experienced transfusion reactions with bolus doses, but no reactions were observed in patients given CI. There were no severe reactions. All the reactions resolved following interruption of the infusion and administration of steroids. Premedication did not prevent reactions. In this series the median decrease in platelet count after bolus injection of pFVIII:C was -67 X 10(9) L(-1) compared with a median decrease of -2 x 109 L(-1) during the course of CI, thus, continuous infusion of pFVIII:C appears to have less effect on platelet count than bolus injection. An anamnestic response was associated with 77% of infusions. This high rate of anamnesis reflects patient selection, in that they were all known to have high-level high-responding FVIII inhibitors with cross-reactivity to pFVIII. After reconstitution, the pFVIII:C showed little loss in factor VIII activity in solution over a 24-h period. We conclude that pFVIII:C may be effectively administered by CI to patients with haemophilia A and high-responding FVIII inhibitors. CI is the probably the mode of administration of choice for intensive replacement therapy with pFVIII.

  15. Novel irreversible epidermal growth factor receptor inhibitors by chemical modulation of the cysteine-trap portion.

    PubMed

    Carmi, Caterina; Cavazzoni, Andrea; Vezzosi, Stefano; Bordi, Fabrizio; Vacondio, Federica; Silva, Claudia; Rivara, Silvia; Lodola, Alessio; Alfieri, Roberta R; La Monica, Silvia; Galetti, Maricla; Ardizzoni, Andrea; Petronini, Pier Giorgio; Mor, Marco

    2010-03-11

    Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATP-competitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group, which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic exploration of the role for the warhead group, introducing different cysteine-trapping fragments at position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups, including epoxyamides (compounds 3-6) and phenoxyacetamides (compounds 7-9), were able to irreversibly inhibit EGFR. In particular, at significant lower concentrations than gefitinib (1), (2R,3R)-N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(piperidin-1-ylmethyl)oxirane-2-carboxamide (6) inhibited EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.

  16. Gentisic Acid, a Compound Associated with Plant Defense and a Metabolite of Aspirin, Heads a New Class of in Vivo Fibroblast Growth Factor Inhibitors*

    PubMed Central

    Fernández, Israel S.; Cuevas, Pedro; Angulo, Javier; López-Navajas, Pilar; Canales-Mayordomo, Ángeles; González-Corrochano, Rocío; Lozano, Rosa M.; Valverde, Serafín; Jiménez-Barbero, Jesús; Romero, Antonio; Giménez-Gallego, Guillermo

    2010-01-01

    Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases. PMID:20145243

  17. Ceramide inhibitor myriocin restores insulin/insulin growth factor signaling for liver remodeling in experimental alcohol-related steatohepatitis

    PubMed Central

    Lizarazo, Diana; Zabala, Valerie; Tong, Ming; Longato, Lisa; de la Monte, Suzanne M.

    2015-01-01

    Background and Aim Alcohol-related liver disease (ALD) is mediated in part by insulin resistance. Attendant dysregulation of lipid metabolism increases accumulation of hepatic ceramides that worsen insulin resistance and compromise the structural and functional integrity of the liver. Insulin and insulin growth factor (IGF) stimulate aspartyl-asparaginyl-β-hydroxylase (AAH), which promotes cell motility needed for structural maintenance and remodeling of the liver. AAH mediates its effects by activating Notch, and in ALD, insulin/IGF signaling, AAH, and Notch are inhibited. Method To test the hypothesis that in ALD, hepatic ceramide load contributes to impairments in insulin, AAH, and Notch signaling, control and chronic ethanol-fed adult Long–Evans rats were treated with myriocin, an inhibitor of serine palmitoyl transferase. Livers were used to assess steatohepatitis, insulin/IGF pathway activation, and expression of AAH–Notch signaling molecules. Results Chronic ethanol-fed rats had steatohepatitis with increased ceramide levels; impairments in signaling through the insulin receptor, insulin receptor substrate, and Akt; and decreased expression of AAH, Notch, Jagged, Hairy–Enhancer of Split-1, hypoxiainducible factor 1α, and proliferating cell nuclear antigen. Myriocin abrogated many of these adverse effects of ethanol, particularly hepatic ceramide accumulation, steatohepatitis, and impairments of insulin signaling through Akt, AAH, and Notch. Conclusions In ALD, the histopathology and impairments in insulin/IGF responsiveness can be substantially resolved by ceramide inhibitor treatments, even in the context of continued chronic ethanol exposure. PMID:23802886

  18. Joannsin, a novel Kunitz-type FXa inhibitor from the venom of Prospirobolus joannsi.

    PubMed

    Luan, Ning; Zhou, Chunling; Li, Pengpeng; Ombati, Rose; Yan, Xiuwen; Mo, Guoxiang; Rong, Mingqiang; Lai, Ren; Duan, Zilei; Zheng, Ruiqiang

    2017-03-09

    The repugnatorial glands of millipedes release various defensive chemical secretions. Although varieties of such defensive secretions have been studied, none of them is protein or peptide. Herein, a novel factor Xa (FXa) inhibitor named joannsin was identified and characterised from repugnatorial glands of Prospirobolus joannsi. Joannsin is composed of 72 amino acid residues including six cysteines, which form three intra-molecular disulfide bridges. It is a member of Kunitz-type protease inhibitor family, members of which are also found in the secretory glands of other arthropods. Recombinant joannsin exhibited remarkable inhibitory activity against trypsin and FXa with a Ki of 182.7 ± 14.6 and 29.5 ± 4.7 nM, respectively. Joannsin showed strong anti-thrombosis functions in vitro and in vivo. Joannsin is the first peptide component in millipede repugnatorial glands to be identified and is a potential candidate and/or template for the development of anti-thrombotic agents. These results also indicated that there is Kunitz-type protease inhibitor toxin in millipede repugnatorial glands as in other arthropods secretory glands.

  19. Brainstem brain-derived neurotrophic factor signaling is required for histone deacetylase inhibitor-induced pain relief.

    PubMed

    Tao, Wenjuan; Chen, Quan; Wang, Lu; Zhou, Wenjie; Wang, Yunping; Zhang, Zhi

    2015-06-01

    Our previous study demonstrated that persistent pain can epigenetically suppress the transcription of Gad2 [encoding glutamic acid decarboxylase 65 (GAD65)] and consequently impair the inhibitory function of GABAergic synapses in central pain-modulating neurons. This contributes to the development of persistent pain sensitization. Histone deacetylase (HDAC) inhibitors increased GAD65 activity considerably, restored GABA synaptic function, and rendered sensitized pain behavior less pronounced. However, the molecular mechanisms by which HDAC regulates GABAergic transmission through GAD65 under pain conditions are unknown. This work showed that HDAC inhibitor-induced increases in colocalization of GAD65 and synaptic protein synapsin I on the presynaptic axon terminals of the nucleus raphe magnus (NRM) were blocked by a TrkB receptor antagonist K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], indicating that BDNF-TrkB signaling may be required in GAD65 modulation of GABA synaptic function. At the brain-derived neurotrophic factor (BDNF) promoter, HDAC inhibitors induced significant increases in H3 hyperacetylation, consistent with the increase in BDNF mRNA and total proteins. Although exogenous BDNF facilitated GABA miniature inhibitory postsynaptic currents and GAD65 accumulation in NRM neuronal synapses in normal rats, it failed to do so in animals subjected to persistent inflammation. In addition, blockade of the TrkB receptor with K252a has no effect on miniature inhibitory postsynaptic currents and synaptic GAD65 accumulation under normal conditions. In addition, the analgesic effects of HDAC inhibitors on behavior were blocked by NRM infusion of K252a. These findings suggest that BDNF-TrkB signaling is required for drugs that reverse the epigenetic effects of chronic pain at the gene level, such as HDAC inhibitors.

  20. Establishing the heparin therapeutic range using aPTT and anti-Xa measurements for monitoring unfractionated heparin therapy

    PubMed Central

    Byun, Jung-Hyun; Jang, In-Seok; Kim, Jong Woo

    2016-01-01

    Background Unfractionated heparin (UFH) has unstable pharmacokinetics and requires close monitoring. The activated partial thromboplastin time (aPTT) test has been used to monitor UFH therapy for decades in Korea, but its results can be affected by numerous variables. We established an aPTT heparin therapeutic range (HTR) corresponding to therapeutic anti-Xa levels for continuous intravenous UFH administration, and used appropriate monitoring to determine if an adequate dose of UFH was applied. Methods A total of 134 ex vivo samples were obtained from 71 patients with a variety of thromboembolisms. All patients received intravenous UFH therapy and were enrolled from June to September 2015 at Gyeongsang National University Hospital. All laboratory protocols were in accordance with the Clinical and Laboratory Standards Institute guidelines and the College of American Pathologist requirements for aPTT HTR. Results An aPTT range of 87.1 sec to 128.7 sec corresponded to anti-Xa levels of 0.3 IU/mL to 0.7 IU/mL for HTR under our laboratory conditions. Based on their anti-Xa levels, blood specimen distribution were as follows: less than 0.3 IU/mL, 65.7%; 0.3–0.7 IU/mL (therapeutic range), 33.6%; and more than 0.7 IU/mL, 0.7%. No evidence of recurring thromboembolism was observed. Conclusion Using the conventional aPTT target range may lead to inappropriate dosing of UFH. Transitioning from the aPTT test to the anti-Xa assay is required to avoid the laborious validation of the aPTT HTR test, even though the anti-Xa assay is more expensive. PMID:27722127

  1. PTT Test

    MedlinePlus

    ... monitor warfarin therapy. Other anticoagulants—anticoagulation therapy with direct thrombin inhibitor (e.g., argatroban, dabigatran) or direct factor Xa inhibitor (e.g., rivaroxaban) Prolonged PTT ...

  2. Recurrent Streptococcus agalactiae Toxic Shock Syndrome Triggered by a Tumor Necrosis FactorInhibitor

    PubMed Central

    Yoshida, Masataka; Takazono, Takahiro; Tashiro, Masato; Saijo, Tomomi; Morinaga, Yoshitomo; Yamamoto, Kazuko; Nakamura, Shigeki; Imamura, Yoshifumi; Miyazaki, Taiga; Sawai, Toyomitsu; Nishino, Tomoya; Izumikawa, Koichi; Yanagihara, Katsunori; Mukae, Hiroshi; Kohno, Shigeru

    2016-01-01

    Streptococcal toxic shock syndrome caused by group B streptococcus (GBS) is a rare, but lethal disease. We experienced a 45-year-old woman with pustular psoriasis who developed toxic shock-like syndrome during infliximab treatment. Surprisingly, similar episodes recurred three times in one year with restarting of infliximab treatments. In the third episode, GBS were detected in blood, urine, and vaginal secretion cultures. These episodes of shock syndrome were possibly due to GBS. To the best of our knowledge, this is the first case report of recurrent streptococcal toxic shock syndrome possibly caused by GBS which was induced by anti-TNF-α inhibitor therapy. The restarting of biological agents in patients with a history of toxic shock syndrome should therefore be avoided as much as possible. PMID:27803422

  3. Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

    PubMed Central

    Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

    2016-01-01

    Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation

  4. Plasma factor and inhibitor composition contributes to thrombin generation dynamics in patients with acute or previous cerebrovascular events

    PubMed Central

    Gissel, Matthew; Undas, Anetta; Slowik, Agnieszka; Mann, Kenneth G.; Brummel-Ziedins, Kathleen E.

    2010-01-01

    Introduction More than 80% of cerebrovascular events are ischemic and largely thromboembolic by nature. We evaluated whether plasma factor composition and thrombin generation dynamics might be a contributor to the thrombotic phenotype of ischemic cerebrovascular events. Materials and Methods We studied (1) 100 patients with acute ischemic stroke (n=50) or transient ischemic attack (TIA) (n=50) within the first 24 hours from symptom onset, and (2) 100 individuals 1 to 4 years following ischemic stroke (n=50) or TIA (n=50). The tissue factor pathway to thrombin generation was simulated with a mathematical model using plasma levels of clotting factors (F)II, V, VII, VIII, IX, X, antithrombin and free tissue factor pathway inhibitor (TFPI). Results The plasma levels of free TFPI, FII, FVIII, and FX were higher, while antithrombin was lower, in the acute patients compared to the previous event group (all p≤0.02). Thrombin generation during acute events was enhanced, with an 11% faster maximum rate, a 15% higher maximum level and a 26% larger total production (all p<0.01). The increased thrombin generation in acute patients was determined by higher FII and lower antithrombin, while increased free TFPI mediated this effect. When the groups are classified by etiology, all stroke sub-types except cardioembolic have increased TFPI and decreased AT and total thrombin produced. Conclusion Augmented thrombin generation in acute stroke/TIA is to some extent determined by altered plasma levels of coagulation factors. PMID:20709367

  5. Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

    PubMed

    Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

    2012-01-01

    Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

  6. Identification of selective covalent inhibitors of platelet activating factor acetylhydrolase 1B2 from the screening of an oxadiazolone-capped peptoid-azapeptoid hybrid library.

    PubMed

    Sarma, Bani Kanta; Liu, Xiaodan; Kodadek, Thomas

    2016-09-01

    A potent and selective inhibitor of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is described. The compound was derived by improvement of a modest affinity primary hit isolated from the screening of a bead-displayed peptoid-azapeptoid hybrid library tethered to an oxadiazolone 'warhead'. The oxadiazolone moiety of the inhibitors was found to react covalently with the active site serine residue of PAFAH1B2. This screening strategy may be useful for the identification of many selective, covalent inhibitors of serine hydrolases.

  7. Trametinib, a novel MEK kinase inhibitor, suppresses lipopolysaccharide-induced tumor necrosis factor (TNF)-α production and endotoxin shock.

    PubMed

    Du, Shi-lin; Yuan, Xue; Zhan, Sun; Tang, Luo-jia; Tong, Chao-yang

    2015-03-13

    Lipopolysaccharide (LPS), one of the most prominent pathogen-associated molecular patterns (PAMPs), activates macrophages, causing release of toxic cytokines (i.e. tumor necrosis factor (TNF)-α) that may provoke inflammation and endotoxin shock. Here, we tested the potential role of trametinib, a novel and highly potent MAPK/ERK kinase (MEK) inhibitor, against LPS-induced TNF-α response in monocytes, and analyzed the underlying mechanisms. We showed that trametinib, at nM concentrations, dramatically inhibited LPS-induced TNF-α mRNA expression and protein secretion in transformed (RAW 264.7 cells) and primary murine macrophages. In ex-vivo cultured human peripheral blood mononuclear cells (PBMCs), this MEK inhibitor similarly suppressed TNF-α production by LPS. For the mechanism study, we found that trametinib blocked LPS-induced MEK-ERK activation in above monocytes, which accounted for the defective TNF-α response. Macrophages or PBMCs treated with a traditional MEK inhibitor PD98059 or infected with MEK1/2-shRNA lentivirus exhibited a similar defect as trametinib, and nullified the activity of trametinib. On the other hand, introducing a constitutively-active (CA) ERK1 restored TNF-α production by LPS in the presence of trametinib. In vivo, mice administrated with trametinib produced low levels of TNF-α after LPS stimulation, and these mice were protected from LPS-induced endotoxin shock. Together, these results show that trametinib inhibits LPS-induced TNF-α expression and endotoxin shock probably through blocking MEK-ERK signaling.

  8. Histone deacetylase inhibitors suppress TF-kappaB-dependent agonist-driven tissue factor expression in endothelial cells and monocytes.

    PubMed

    Wang, Jianguo; Mahmud, Shawn A; Bitterman, Peter B; Huo, Yuqing; Slungaard, Arne

    2007-09-28

    Histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA), can regulate gene expression by promoting acetylation of histones and transcription factors. Human tissue factor (TF) expression is partly governed by a unique, NF-kappaB-related "TF-kappaB" promoter binding site. We find that TSA and four other HDACi (apicidin, MS-275, sodium butyrate, and valproic acid) all inhibit by approximately 90% TF activity and protein level induction in human umbilical vein endothelial cells stimulated by the physiologic agonists tumor necrosis factor (TNF)-alpha, interleukin-1beta, lipopolysaccharide, and HOSCN without affecting expression of the NF-kappaB-regulated adhesion molecules ICAM-1 and E-selectin. TSA and butyrate also blunt TF induction approximately 50% in vitro in peripheral blood mononuclear cells and in vivo in thioglycolate-elicited murine peritoneal macrophages. In human umbilical vein endothelial cells, TSA attenuates by approximately 70% TNF-alpha stimulation of TF mRNA transcription without affecting that of ICAM-1. By electrophoretic mobility shift assay analyses, TNF-alpha and lipopolysaccharide induce strong p65/p50 and p65/c-Rel heterodimer binding to both NF-kappaB and TF-kappaB probes. TSA nearly abolishes TF-kappaB binding without affecting NF-kappaB binding. A chromatin immunoprecipitation assay and a promoter-luciferase reporter system confirm that TSA inhibits TF-kappaB but not NF-kappaB activation. Chromatin immunoprecipitation and small interfering RNA inhibitor studies demonstrate that HDAC3 plays a significant role in TNF-alpha-mediated TF induction. Thus, HDACi transcriptionally inhibit agonist-induced TF expression in endothelial cells and monocytes by a TF-kappaB- and HDAC3-dependent mechanism. We conclude that histone deacetylases, particularly HDAC3, play a hitherto unsuspected role in regulating TF expression and raise the possibility that HDACi might be a novel therapy for thrombotic disorders.

  9. Melasma treatment: A novel approach using a topical agent that contains an anti-estrogen and a vascular endothelial growth factor inhibitor.

    PubMed

    Cohen, Philip R

    2017-04-01

    Melasma is an acquired disorder of pigmentation that presents with asymptomatic symmetric darkening of the face. The pathogenesis of this condition is multifactorial and influenced by several factors including female sex hormones, genetic predisposition and ultraviolet light exposure. The management of melasma is usually directed at more than one of the causative etiologic factors and often incorporates a combination of topical agents, with or without the addition of physical modalities. Estrogen and angiogenesis are significant factors in the etiology of melasma. A useful addition to the therapeutic armentarium for treating melasma would include a topical agent that could effect both of these causative factors. Specifically, a topical preparation consisting of an anti-estrogen and a vascular endothelial growth factor inhibitor would accomplish this goal. Suitable candidates that target estrogen receptors and vascular endothelial growth factor are currently used in medical oncology as systemic antineoplastic agents. The anti-estrogen could be either a selective estrogen receptor modulator (such as tamoxifen or raloxifene) or an aromatase inhibitor (such as anastrozole or letrozole or exemestane). The vascular endothelial growth factor inhibitor would be bevacizumab. In conclusion, a novel-topically administered-therapy for melasma would combine an anti-estrogen and a vascular endothelial growth factor inhibitor.

  10. Stimulation of tumor necrosis factor alpha production in human monocytes by inhibitors of protein phosphatase 1 and 2A

    PubMed Central

    1992-01-01

    The protein phosphatase 1 and 2A inhibitor, okadaic acid, has been shown to stimulate many cellular functions by increasing the phosphorylation state of phosphoproteins. In human monocytes, okadaic acid by itself stimulates tumor necrosis factor alpha (TNF-alpha) mRNA accumulation and TNF-alpha synthesis. Calyculin A, a more potent inhibitor of phosphatase 1, has similar effects. TNF-alpha mRNA accumulation in okadaic acid-treated monocytes is due to increased TNF- alpha mRNA stability and transcription rate. The increase in TNF-alpha mRNA stability is more remarkable in okadaic acid-treated monocytes than the mRNA stability of other cytokines, such as interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6. Gel retardation studies show the stimulation of AP-1, AP-2, and NF-kappa B binding activities in okadaic acid-stimulated monocytes. This increase may correlate with the increase in TNF-alpha mRNA transcription rate. In addition to the stimulation of TNF-alpha secretion by monocytes, okadaic acid appears to modulate TNF-alpha precursor processing, as indicated by a marked increase in the cell-associated 26-kD precursor. These results suggest that active basal phosphorylation/dephosphorylation occurs in monocytes, and that protein phosphatase 1 or 2A is important in regulating TNF-alpha gene transcription, translation, and posttranslational modification. PMID:1324971

  11. Peroxisome Proliferator-Activated Receptor γ Induces the Expression of Tissue Factor Pathway Inhibitor-1 (TFPI-1) in Human Macrophages

    PubMed Central

    Copin, C.; Derudas, B.; Marx, N.

    2016-01-01

    Tissue factor (TF) is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa). Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1). Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that, together with PPARα and PPARβ/δ, controls macrophage functions. However, whether PPARγ activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPARγ activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPARγ ligands, an effect shared by the activation of PPARα and PPARβ/δ, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPARγ ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPARγ in the control of TF the pathway. PMID:28115923

  12. Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli.

    PubMed

    Bønsager, Birgit C; Praetorius-Ibba, Mette; Nielsen, Peter K; Svensson, Birte

    2003-08-01

    Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.

  13. Factor XI and XII as antithrombotic targets

    PubMed Central

    Müller, Felicitas; Gailani, David; Renné, Thomas

    2015-01-01

    Purpose of review Arterial and venous thrombosis are major causes of morbidity and mortality, and the incidence of thromboembolic diseases increases as a population ages. Thrombi are formed by activated platelets and fibrin. The latter is a product of the plasma coagulation system. Currently available anticoagulants such as heparins, vitamin K antagonists and inhibitors of thrombin or factor Xa target enzymes of the coagulation cascade that are critical for fibrin formation. However, fibrin is also necessary for terminating blood loss at sites of vascular injury. As a result, anticoagulants currently in clinical use increase the risk of bleeding, partially offsetting the benefits of reduced thrombosis. This review focuses on new targets for anticoagulation that are associated with minimal or no therapy-associated increased bleeding. Recent findings Data from experimental models using mice and clinical studies of patients with hereditary deficiencies of coagulation factors XI or XII have shown that both of these clotting factors are important for thrombosis, while having minor or no apparent roles in processes that terminate blood loss (hemostasis). Summary Hereditary deficiency of factor XII (Hageman factor) or factor XI, plasma proteases that initiate the intrinsic pathway of coagulation, impairs thrombus formation and provides protection from vascular occlusive events, while having a minimal impact on hemostasis. As the factor XII–factor XI pathway contributes to thrombus formation to a greater extent than to normal hemostasis, pharmacological inhibition of these coagulation factors may offer the exciting possibility of anticoagulation therapies with minimal or no bleeding risk. PMID:21730835

  14. Role of vascular endothelial growth factor inhibitors in the treatment of gynecologic malignancies.

    PubMed

    Burger, Robert A

    2010-03-01

    This article reviews the history and current status of vascular endothelial growth factor targeted therapy for the most common gynecologic malignancies - epithelial ovarian, endometrial and cervical cancers. The biologic rationale for targeting vascular endothelial growth factor (VEGF) for these disease sites is well-founded, and pre-clinical studies have supported the development of anti-VEGF agents. Their classification, known mechanisms of action, unique toxicities and clinical development are herein explored, the latter including issues related to study design, disease site and disease setting.

  15. Prefoldin Plays a Role as a Clearance Factor in Preventing Proteasome Inhibitor-induced Protein Aggregation*

    PubMed Central

    Abe, Akira; Takahashi-Niki, Kazuko; Takekoshi, Yuka; Shimizu, Takashi; Kitaura, Hirotake; Maita, Hiroshi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2013-01-01

    Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases. PMID:23946485

  16. Neutrophil biomarkers predict response to therapy with tumor necrosis factor inhibitors in rheumatoid arthritis.

    PubMed

    Wright, Helen L; Cox, Trevor; Moots, Robert J; Edwards, Steven W

    2017-03-01

    Neutrophils are implicated in the pathology of rheumatoid arthritis (RA), but the mechanisms regulating their activation are largely unknown. RA is a heterogeneous disease, and whereas many patients show clinical improvement during TNF inhibitor (TNFi) therapy, a significant proportion fails to respond. In vitro activation of neutrophils with agents, including TNF, results in rapid and selective changes in gene expression, but how neutrophils contribute to TNF signaling in RA and whether TNFi sensitivity involves differential neutrophil responses are unknown. With the use of RNA sequencing (RNA-Seq), we analyzed blood neutrophils from 20 RA patients, pre-TNFi therapy, to identify biomarkers of response, measured by a decrease in disease activity score based on 28 joint count (DAS28), 12 wk post-therapy. Biomarkers were validated by quantitative PCR (qPCR) of blood neutrophils from 2 further independent cohorts of RA patients: 16 pre-TNFi and 16 predisease-modifying anti-rheumatic drugs (DMARDs). Twenty-three neutrophil transcripts predicted a 12-wk response to TNFi: 10 (IFN-regulated) genes predicting a European League against Rheumatism (EULAR) good response and 13 different genes [neutrophil granule protein (NGP) genes] predicting a nonresponse. Statistical analysis indicated a predictive sensitivity and specificity of each gene in the panel of >80%, with some 100% specific. A combination of 3 genes [cytidine monophosphate kinase 2 (CMPK2), IFN-induced protein with tetratricopeptide repeats 1B (IFIT1B), and RNASE3] had the greatest predictive power [area under the curve (AUC) 0.94]. No correlation was found for a response to DMARDs. We conclude that this panel of genes is selective for predicting a response to TNFi and is not a surrogate marker for disease improvement. We also show that in RA, there is great plasticity in neutrophil phenotype, with circulating cells expressing genes normally only expressed in more immature cells.

  17. A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma.

    PubMed

    Wang, Liqin; Šuštić, Tonći; Leite de Oliveira, Rodrigo; Lieftink, Cor; Halonen, Pasi; van de Ven, Marieke; Beijersbergen, Roderick L; van den Heuvel, Michel M; Bernards, René; van der Heijden, Michiel S

    2017-01-17

    Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.

  18. Desensitization and immune tolerance induction in children with severe factor IX deficiency; inhibitors and adverse reactions to replacement therapy: a case-report and literature review.

    PubMed

    Bon, Andrea; Morfini, Massimo; Dini, Alessandro; Mori, Francesca; Barni, Simona; Gianluca, Sottilotta; de Martino, Maurizio; Novembre, Elio

    2015-02-19

    Hemophilia B is a rare X-linked recessive disorder with plasma factor IX (FIX) deficiency. 1-3% of patients treated with exogenous FIX-containing products develop inhibitors (i.e. polyclonal high affinity immunoglobulins) that neutralize the procoagulant activity of a specific coagulation factor. Although the incidence of inhibitors in hemophilia B patients is low, most are "high titer" and frequently associated with the development of severe allergic or anaphylactic reactions. Immune tolerance induction as a strategy for inhibitor eradication was first described in 1984. Unfortunately, the overall reported success of immune tolerance induction in FIX deficiency with inhibitors is approximately 25-40%.We report the case of a 2-year-old boy with hemophilia B severe FIX deficiency (<1%), inhibitor antibodies to FIX development, and a history of adverse reactions to FIX infusions, who underwent a successful desensitization and immune tolerance induction with a daily FIX infusion. With this regimen the inhibitor titer decreased with effective bleeding prevention.

  19. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    PubMed

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  20. Is there a role for epidermal growth factor receptor tyrosine kinase inhibitors in epidermal growth factor receptor wild-type non-small cell lung cancer?

    PubMed Central

    Arriola, Edurne; Taus, Álvaro; Casadevall, David

    2015-01-01

    Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a world-wide annual incidence of around 1.3 million. The majority of patients are diagnosed with advanced disease and survival remains poor. However, relevant advances have occurred in recent years through the identification of biomarkers that predict for benefit of therapeutic agents. This is exemplified by the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors for the treatment of EGFR mutant patients. These drugs have also shown efficacy in unselected populations but this point remains controversial. Here we have reviewed the clinical data that demonstrate a small but consistent subgroup of EGFR wild-type patients with NSCLC that obtain a clinical benefit from these drugs. Moreover, we review the biological rationale that may explain this benefit observed in the clinical setting. PMID:26266101

  1. A low balance between microparticles expressing tissue factor pathway inhibitor and tissue factor is associated with thrombosis in Behçet’s Syndrome

    PubMed Central

    Khan, E.; Ambrose, N. L.; Ahnström, J.; Kiprianos, A. P.; Stanford, M. R.; Eleftheriou, D.; Brogan, P.A.; Mason, J. C.; Johns, M.; Laffan, M. A.; Haskard, D. O.

    2016-01-01

    Thrombosis is common in Behçet’s Syndrome (BS), and there is a need for better biomarkers for risk assessment. As microparticles expressing Tissue Factor (TF) can contribute to thrombosis in preclinical models, we investigated whether plasma microparticles expressing Tissue Factor (TF) are increased in BS. We compared blood plasma from 72 healthy controls with that from 88 BS patients (21 with a history of thrombosis (Th+) and 67 without (Th−). Using flow cytometry, we found that the total plasma MP numbers were increased in BS compared to HC, as were MPs expressing TF and Tissue Factor Pathway Inhibitor (TFPI) (all p < 0.0001). Amongst BS patients, the Th+ group had increased total and TF positive MP numbers (both p ≤ 0.0002) compared to the Th- group, but had a lower proportion of TFPI positive MPs (p < 0.05). Consequently, the ratio of TFPI positive to TF positive MP counts (TFPI/TF) was significantly lower in Th+ versus Th− BS patients (p = 0.0002), and no patient with a TFPI/TF MP ratio >0.7 had a history of clinical thrombosis. We conclude that TF-expressing MP are increased in BS and that an imbalance between microparticulate TF and TFPI may predispose to thrombosis. PMID:27924945

  2. Successful treatment of a spontaneous haemothorax with recombinant factor VIIa in a haemophilic child with inhibitors

    PubMed Central

    Gidaris, D; Economou, M; Valeri, R; Gombakis, N; Athanasiou - Metaxa, M

    2010-01-01

    Haemophilia A is an X-linked disorder caused by a deficiency of factor VIII. Haemorrhage in various sites may occur spontaneously or secondary to trauma depending on the severity of the deficiency. Common manifestations include haemarthrosis, epistaxis, gastrointestinal haemorrhage and haematuria. Spontaneous haemothorax has rarely been reported both in children and adults1,2. We report the case of a haemophiliac child presenting with spontaneous haemothorax due to the rarity of this clinical presentation in order to raise the awareness among clinicians. PMID:21311642

  3. Effects of nuclear factor-κB and ERK signaling transduction pathway inhibitors on human melanoma cell proliferation in vitro.

    PubMed

    Huang, Yi-Chuan; Pan, Min; Liu, Ning; Xiao, Jun-Gang; Chen, Hong-Quan

    2015-11-01

    The present study aimed to investigate the effects of blocking nuclear factor (NF)-κB and/or extracellular signal-regulated kinase (ERK) signaling pathways on proliferation and apoptosis of melanoma cells in vitro. A375 Human melanoma cells were treated with U0126 (ERK signaling pathway inhibitor) and BMS-345541 (NF-κB inhibitor), alone or in combination. At 12, 24 and 48 h after treatment, cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle progression and apoptosis were evaluated by flow cytometry, and Bcl-2 protein content was determined by western blot analysis. BMS-345541 and U0126 significantly inhibited A375 cell proliferation in a dose- and time-dependent manner (P<0.01). The rate of proliferation inhibition at 24 h was 35.41±1.38% for BMS-345541 alone, 30.64±2.86% for U0126 alone, and 77.27±2.70% for BMS-345541 and U0126 in combination. The difference between combination and single treatment was significantly different (P<0.01). The proportion of cells in S phase was 14.20, 18.40 and 22.64% following treatment with BMS-345541, U0126, and BMS-345541 and U0126 in combination, respectively; these values were all significantly reduced compared with the untreated control group (P<0.01). The apoptosis rate was 24.98±1.03% in the BMS-345541 group, 13.96±0.96% in the U0126 group and 38.91±1.46% in the combination group; all significantly increased compared with the control group (P<0.01). Bcl-2 protein content in A375 cells was significantly increased following treatment with BMS-345541 and U0126, alone or in combination, when compared with the untreated control group (P<0.01). Therefore, NF-κB and ERK signaling pathway inhibitors may serve as potential therapeutic targets for melanoma.

  4. Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation

    SciTech Connect

    Rancourt, Raymond C. Veress, Livia A. Ahmad, Aftab Hendry-Hofer, Tara B. Rioux, Jacqueline S. Garlick, Rhonda B. White, Carl W.

    2013-10-01

    Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. Methods: Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5 mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O{sub 2} saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin–antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. Results: Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. Conclusions: Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury. - Highlights: • TFPI administration to rats after mustard inhalation reduces airway cast formation. • Inhibition of thrombin activation is the likely mechanism for limiting casts. • Rats given TFPI

  5. Alpha-1 proteinase inhibitor M358R reduces thrombin generation when displayed on the surface of cells expressing tissue factor.

    PubMed

    Gierczak, Richard F; Pepler, Laura; Bhagirath, Vinai; Liaw, Patricia C; Sheffield, William P

    2014-11-01

    The M358R variant of alpha-1-proteinase inhibitor (API) is a potent soluble inhibitor of thrombin. Previously we engineered AR-API M358R, a membrane-bound form of this protein and showed that it inhibited exogenous thrombin when expressed on transfected cells lacking tissue factor (TF). To determine the suitability of AR-API M358R for gene transfer to vascular cells to limit thrombogenicity, we tested the ability of AR-API M358R to inhibit endogenous thrombin generated in plasma via co-expression co-expressing it on the surface of cells expressing TF. Transfected AR-API M358R formed inhibitory complexes with thrombin following exposure of recalcified, defibrinated plasma to TF on T24/83 cells, but discontinuously monitored thrombin generation was unaffected. Similarly, AR-API M358R expression did not reduce continuously monitored thrombin generation by T24/83 cell suspensions exposed to recalcified normal plasma in a Thrombogram-Thrombinoscope-type thrombin generation assay (TGA); in contrast, 1 μM hirudin variant 3 or soluble API M358R abolished thrombin generation. Gene transfer of TF to HEK 293 conferred the ability to support TF-dependent thrombin generation on HEK 293 cells. Co-transfection of HEK 293 cells with a 9:1 excess of DNA encoding AR-API M358R to that encoding TF reduced peak thrombin generation approximately 3-fold compared to controls. These in vitro results suggest that surface display of API M358R inhibits thrombin generation when the tethered serpin is expressed in excess of TF, and suggest its potential to limit thrombosis in appropriate vascular beds in animal models.

  6. Secondary prophylaxis with recombinant activated factor VII improves health-related quality of life of haemophilia patients with inhibitors.

    PubMed

    Hoots, W K; Ebbesen, L S; Konkle, B A; Auerswald, G K-H; Roberts, H R; Weatherall, J; Ferran, J-M; Ljung, R C R

    2008-05-01

    Haemophilia patients with inhibitors characteristically have impaired joint function and reduced health-related quality of life (HRQoL). This analysis examined whether secondary prophylaxis with recombinant activated factor VII (rFVIIa) improves HRQoL vs. conventional on-demand therapy in patients with haemophilia with inhibitors and frequent bleeds. After a 3-month preprophylaxis period, 22 patients received daily rFVIIa prophylaxis (90 or 270 microg kg(-1)) for 3 months, followed by 3 months' postprophylaxis. Days of hospitalization, absence from school/work and mobility aids requirements were recorded. HRQoL was assessed by EuroQoL (EQ-5D) questionnaire, visual analogue scale (VAS), derived Time to Trade-Off (TTO) scores and Quality Adjusted Life Years (QALYs). rFVIIa prophylaxis significantly (P < 0.0001) reduced bleeding frequency vs. prior on-demand therapy. Hospitalization (5.9% vs. 13.5%; P = 0.0026) and absenteeism from school/work (16.7% vs. 38.7%; P = 0.0127) decreased during prophylaxis; these effects tended to be maintained during postprophylaxis. HRQoL (evaluated by EQ-5D) tended to improve during and after rFVIIa prophylaxis. Notably, pain decreased and mobility increased in 40.9% and 27.3% of patients, respectively, at the end of the postprophylaxis period vs. preprophylaxis. Median VAS score increased from 66 to 73 (P = 0.048), and TTO scores suggested better HRQoL (0.62 vs. 0.76; P = 0.054) during postprophylaxis than preprophylaxis. Small to moderate changes in effect sizes were reported for VAS and TTO scores. Median QALYs were 0.68 (VAS) and 0.73 (TTO). Reductions in bleeding frequency with secondary rFVIIa prophylaxis were associated with improved HRQoL vs. on-demand therapy.

  7. In vitro hepatic metabolism of cediranib, a potent vascular endothelial growth factor tyrosine kinase inhibitor: interspecies comparison and human enzymology.

    PubMed

    Schulz-Utermoehl, Timothy; Spear, Michael; Pollard, Christopher R J; Pattison, Christine; Rollison, Helen; Sarda, Sunil; Ward, Michelle; Bushby, Nick; Jordan, Angela; Harrison, Mike

    2010-10-01

    The in vitro metabolism of cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors in late-stage development for the treatment of colorectal cancer and recurrent glioblastoma was investigated in hepatic proteins from preclinical species and humans using radiolabeled material. In human hepatocyte cultures, oxidative and conjugative metabolic pathways were identified, with pyrrolidine N(+)-glucuronidation being the major route. The primary oxidative pathways were di-and trioxidations and pyrrolidine N-oxidation. All metabolites with the exception of the N(+)-glucuronide metabolite were observed in rat and cynomolgus monkey hepatocyte preparations. Additional metabolism studies in liver microsomes from these or other preclinical species (CD-1 mouse, Han Wistar rat, Dunkin Hartley guinea pig, Göttingen mini-pig, New Zealand White rabbit, beagle dog, and cynomolgus and rhesus monkey) indicated that the N(+)-glucuronide metabolite was not formed in these additional species. Incubations with recombinant flavin-containing monooxygenase (FMO) and UDP-glucuronosyltransferase (UGT) enzymes and inhibition studies using the nonselective cytochrome P450 (P450) chemical inhibitor 1-aminobenzotriazole in human hepatocytes indicated that FMO1 and FMO3 contributed to cediranib N-oxidation, whereas UGT1A4 had a major role in cediranib N(+)-glucuronidation. P450 enzymes had only a minor role in the metabolism of cediranib. In conclusion, species differences in the formation of the N(+)-glucuronide metabolite of cediranib were observed. All other metabolites of cediranib found in humans were also detected in rat and cynomolgus monkey. Non-P450 enzymes are predominantly involved in the metabolism of cediranib, and this suggests that clinical drug interactions involving other coadministered drugs are unlikely.

  8. The broadly effective recessive resistance gene xa5 of rice is a virulence effector-dependent quantitative trait for bacterial blight.

    PubMed

    Huang, Sheng; Antony, Ginny; Li, Ting; Liu, Bo; Obasa, Ken; Yang, Bing; White, Frank F

    2016-04-01

    Mutations in disease susceptibility (S) genes, here referred to as recessive resistance genes, have promise for providing broad durable resistance in crop species. However, few recessive disease resistance genes have been characterized. Here, we show that the broadly effective resistance gene xa5,for resistance to bacterial blight of rice (Oryza sativa), is dependent on the effector genes present in the pathogen. Specifically, the effectiveness of xa5 in preventing disease by strains of Xanthomonas oryzae pv. oryzae is dependent on major transcription activation-like (TAL) effector genes, and correlates with reduced expression of the cognate S genes. xa5 is ineffective in preventing disease by strains containing the TAL effector gene pthXo1, which directs robust expression of the S gene OsSWEET11, a member of sucrose transporter gene family. Incompatibility is associated with major TAL effectors that target the known alternative S genes OsSWEET14 and OsSWEET13. Incompatibility is defeated by transfer of pthXo1 to otherwise xa5-incompatible strains or by engineering a synthetic designer TAL effector to boost SWEET gene expression. In either case, compatible or incompatible, target gene expression and lesion formation are reduced in the presence of xa5. The results indicate that xa5 functions as a quantitative trait locus, dampening effector function, and, regardless of compatibility, target gene expression. Resistance is hypothesized to occur when S gene expression, and, by inference, sucrose leakage, falls below a threshold level.

  9. Activity of fibroblast growth factor receptor inhibitors TKI258, ponatinib and AZD4547 against TPR-FGFR1 fusion

    PubMed Central

    Qiu, Xu-Hua; Li, Feng; Cao, Hong-Qin; Shao, Jing-Jing; Mei, Jian-Gang; Li, Han-Qing; Zhai, Yong-Ping

    2017-01-01

    8p11 myeloproliferative syndrome (EMS) is a rare disease characterized by the constitutive activation of fibroblast growth factor receptor 1 (FGFR1). To date, four cases of EMS with the chromosomal translocation, t(1;8)(q25;p11.2), have been reported. In the present study, TPR-FGFR1-expressing Baf3 cells were established and confirmed by polymerase chain reaction. To identify the most promising drug for EMS, the activities and associated mechanism of three tyrosine kinase inhibitors (TKIs), TKI258, ponatinib and AZD4547, against TPR-FGFR1 were tested by MTT assay, flow cytometry and western blot. The data demonstrated that TPR-FGFR1 was localized in the cytoplasm, and was able to transform interleukin-3-dependent hematopoietic Baf3 cells into growth factor-independent cells. All of the three TKIs markedly inhibited the proliferation of TPR-FGFR1-expressing Baf3 cells, and the activation of FGFR1 and the downstream signaling molecules, extracellular signal-regulated kinase 1/2, phospholipiase Cγ and signal transducer and activator of transcription 5. AZD4547 was the most efficient drug, and TKI258 was the least. By contrast, no significant difference was found among the three drugs on their effect on cell apoptosis. Taken together, the data obtained in the present study suggested that AZD4547 had increased potency, compared with TKI258 and ponatinib, for the treatment of EMS. PMID:28138694

  10. Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

    PubMed

    Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

    2013-08-23

    PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment.

  11. Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis.

    PubMed

    Koehler, Angela N; Shamji, Alykhan F; Schreiber, Stuart L

    2003-07-16

    Small molecule microarrays were screened to identify a small molecule ligand for Hap3p, a subunit of the yeast Hap2/3/4/5p transcription factor complex. The compound, named haptamide A, was determined to have a KD of 5.03 muM for binding to Hap3p using surface plasmon resonance analysis. Haptamide A also inhibited activation of a GDH1-lacZ reporter gene in a dose-dependent fashion. To explore structure-activity relationships, 11 derivatives of haptamide A were prepared using the same synthetic route that was developed for the original library synthesis. Analysis of dissociation constants and IC50 values for the reporter gene assay revealed a more potent inhibitor, haptamide B, with a KD of 330 nM. Whole-genome transcriptional profiling was used to compare effects of haptamide B with a hap3Delta yeast strain. Treatment with haptamide B, like the deletion mutant, reduced lactate-induced transcription of several genes from wild-type levels. Profiling the genetic "knockout" and the chemical genetic "knockdown" led to the identification of several genes that are regulated by Hap3p under nonfermentative conditions. These results demonstrate that a small molecule discovered using the small molecule microarray binding assay can permeate yeast cells and reach its target transcription factor protein in cells.

  12. Virtual Screening of Specific Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors from the National Cancer Institute (NCI) Molecular Database

    PubMed Central

    Fan, Cong; Huang, Yan-Xin; Bao, Yong-Li; Sun, Lu-Guo; Wu, Yin; Yu, Chun-Lei; Zhang, Yu; Song, Zhen-Bo; Zheng, Li-Hua; Sun, Ying; Wang, Guan-Nan; Li, Yu-Xin

    2012-01-01

    Insulin-like growth factor 1 receptor (IGF1R) is an attractive drug target for cancer therapy and research on IGF1R inhibitors has had success in clinical trials. A particular challenge in the development of specific IGF1R inhibitors is interference from insulin receptor (IR), which has a nearly identical sequence. A few potent inhibitors that are selective for IGF1R have been discovered experimentally with the aid of computational methods. However, studies on the rapid identification of IGF1R-selective inhibitors using virtual screening and confidence-level inspections of ligands that show different interactions with IGF1R and IR in docking analysis are rare. In this study, we established virtual screening and binding-mode prediction workflows based on benchmark results of IGF1R and several kinase receptors with IGF1R-like structures. We used comprehensive analysis of the known complexes of IGF1R and IR with their binding ligands to screen specific IGF1R inhibitors. Using these workflows, 17 of 139,735 compounds in the NCI (National Cancer Institute) database were identified as potential specific inhibitors of IGF1R. Calculations of the potential of mean force (PMF) with GROMACS were further conducted for three of the identified compounds to assess their binding affinity differences towards IGF1R and IR. PMID:23242155

  13. How useful is the monitoring of (low molecular weight) heparin therapy by anti-Xa assay? A laboratory perspective.

    PubMed

    Favaloro, Emmanuel J; Bonar, Roslyn; Aboud, Margaret; Low, Joyce; Sioufi, John; Wheeler, Michael; Lloyd, John; Street, Alison; Marsden, Katherine

    2005-01-01

    We have conducted a series of laboratory-based surveys to assess variability in assay results utilized to monitor heparin anticoagulant therapy. These surveys involved laboratories participating in the Haematology component of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP). Thirty five of 646 laboratories that were sent a preliminary questionnaire indicated that they performed anti-Xa assays and these laboratories were sent a panel of four plasma samples. These plasma samples contained respectively: (i) no added heparin, (ii) low molecular weight heparin (LMWH), enoxaparin, added to a level of approximately .5 U/mL, (iii) unfractionated heparin added to a level of approximately .5 U/mL, and (iv) LMWH added to a level of approximately 1.0 U/mL. Tests to be performed were the activated partial thromboplastin time (APTT), the thrombin time (TT), fibrinogen, and anti-Xa. As expected, returned results for APTT and TT showed some elevation in heparinized samples while fibrinogen assays were not affected. Anti-Xa assays yielded the following results (median [range]): (i) .01 [0-.11], (ii) .43 [.33-.80], (iii) .23 [.10-.49], and (iv) .90 [.60-1.30]. Thus, although median values were close to those anticipated, there was a wide variation in returned results. In a repeat exercise a few months later laboratories were also asked about their therapeutic ranges (TRs) and provided with an additional vial of LMWH-spiked (1.0 U/mL) plasma labeled as 'heparin-standard' to be used as an assay calibrant. TRs varied substantially between laboratories, from low ranges of .2-.4 to high ranges of .8-1.2. Anti-Xa assay results were similar to those of the first survey: (median [range]): (a) repeat testing: (i) .02 [0-.28], (ii) .47 [.34-.80], (iii) .25 [.14-.58], (iv) .95 [.65-1.31]; (b) repeat testing using survey provided 'heparin-standard': (i) .02 [0-.24], (ii) .55 [.4-.83], (iii) .28 [.10-.63], (iv) 1.00 [.9-1.16]. Thus using the provided

  14. Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    SciTech Connect

    VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom,Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor,Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.

    2005-09-01

    The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.

  15. Assessment of the frequency of regulatory T cells (CD4+CD25+CD127-) in children with hemophilia A: relation to factor VIII inhibitors and disease severity.

    PubMed

    El-Asrar, Mohamed Abo; Hamed, Ahmed El-Saeed; Darwish, Yasser Wagih; Ismail, Eman Abdel Rahman; Ismail, Noha Ali

    2016-01-01

    A rapidly growing evidence showed that regulatory T cells (Tregs) play a crucial role in tolerance to coagulation factors and may be involved in the pathogenesis of inhibitor formation in patients with hemophilia. We determined the percentage of Tregs (CD4CD25CD127) in 45 children with hemophilia A compared with 45 healthy controls, and assessed their relation to the clinical characteristics of patients and factor VIII (FVIII) inhibitors. Patients were studied stressing on frequency of bleeding attacks, joint pain, history of viral hepatitis, and the received therapy (FVIII precipitate/cryotherapy). FVIII activity and FVIII inhibitors were assessed with flow cytometric analysis of CD4CD25CD127 Tregs. According to residual FVIII activity levels, 30 patients (66.7%) had mild/moderate hemophilia A, whereas 15 (33.3%) patients had severe hemophilia A. The frequency of Tregs was significantly lower among all patients with hemophilia A compared with controls (2.59 ± 1.1 versus 3.73 ± 1.12%; P = 0.002). Tregs were significantly decreased among patients with FVIII inhibitors compared with the inhibitor-negative group (P < 0.001). Patients with hematuria or severe hemophilia A had lower Tregs levels than those without (P = 0.34 and P = 0.011, respectively). A significant positive correlation was found between the percentage of Tregs and FVIII among hemophilia A patients. ROC curve analysis revealed that the cut-off value of Tregs at 1.91% could differentiate patients with and without FVIII inhibitors, with a sensitivity of 100% and a specificity of 91.3%. We suggest that alteration in the frequency of Tregs in young patients with hemophilia A may contribute to inhibitor formation and disease severity.

  16. Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

    PubMed

    Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

    2017-01-01

    Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer.

  17. CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis

    PubMed Central

    Citro, Alessandra; Scrivo, Rossana; Martini, Helene; Martire, Carmela; De Marzio, Paolo; Vestri, Anna Rita; Sidney, John; Sette, Alessandro; Barnaba, Vincenzo; Valesini, Guido

    2015-01-01

    CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes) represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factorinhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control). The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factorinhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factorinhibitor therapy to validate in a larger cohort of patients. PMID:26061065

  18. Risk Factors for Nursing Home Placement in Alzheimer's Disease: A Longitudinal Study of Cognition, ADL, Service Utilization, and Cholinesterase Inhibitor Treatment

    ERIC Educational Resources Information Center

    Wattmo, Carina; Wallin, Asa K.; Londos, Elisabet; Minthon, Lennart

    2011-01-01

    Purpose of the Study: To identify risk factors for early nursing home placement (NHP) in Alzheimer's disease (AD), focusing on the impact of longitudinal change in cognition, activities of daily living (ADL), service utilization, and cholinesterase inhibitor treatment (ChEI). Design and Methods: In an open, 3-year, prospective, multicenter study…

  19. Activation of factor X by rat hepatocytes

    SciTech Connect

    Willingham, A.K.; Matschiner, J.T.

    1986-05-01

    Synthesis and secretion of blood coagulation factor X was studied in hepatocytes prepared by perfusion of rat livers with collagenase. Hepatocytes were incubated in the presence of vitamin K and /sup 3/H-leucine for up to 4h at 37/sup 0/C. Factor X was isolated from the incubation medium by immunochemical techniques and analyzed by SDS-PAGE. The recovered /sup 3/H-labeled proteins migrated, after reduction of disulfides, as two polypeptide chains with apparent molecular weights (M/sub r/) of approximately 42,000 and 22,000 representing the heavy and light chains of factor X respectively. The apparent M/sub r/ of the heavy chain was about 10,000 daltons lighter than seen with the heavy chain of factor X isolated from rat plasma and was more characteristic of the heavy chain of factor Xa. When the levels of factor X secreted by hepatocytes were determined by clotting assays, activity was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes (>95% parenchymal cells) the added factor X was rapidly converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium dependent reaction. The physiological significance of a factor X activating enzyme on hepatocyte plasma membranes is not clear.

  20. Tumor necrosis factor inhibitor therapy in ankylosing spondylitis: differential effects on pain and fatigue and brain correlates.

    PubMed

    Wu, Qi; Inman, Robert D; Davis, Karen D

    2015-02-01

    Ankylosing spondylitis is associated with back pain and fatigue and impacts mobility but can be treated with tumor necrosis factor inhibitors (TNFi). The differential effects of TNFi treatment on multiple symptoms and the brain is not well delineated. Thus, we conducted a 2-part study. In study 1, we conducted a retrospective chart review in 129 ankylosing spondylitis patients to assess TNFi effects on pain, fatigue, motor function, mobility, and quality of life (QoL). After at least 10 weeks of TNFi treatment, patients had clinically significant improvements (>30%) in pain (including neuropathic pain), most disease and QoL factors, and normalized sensory detection thresholds. However, residual fatigue (mean = 5.3) was prominent. Although 60% of patients had significant relief of pain, only 22% of patients had significant relief of both pain and fatigue. Therefore, the preferential TNFi treatment effect on pain compared with fatigue could contribute to suboptimal effects on QoL. Part 2 was a prospective study in 14 patients to identify TNFi treatment effects on pain, fatigue, sensory and psychological factors, and brain cortical thickness based on 3T magnetic resonance imaging. Centrally, TNFi was associated with statistically significant cortical thinning of motor, premotor, and posterior parietal regions. Pain intensity reduction was associated with cortical thinning of the secondary somatosensory cortex, and pain unpleasantness reduction was associated with the cortical thinning of motor areas. In contrast, fatigue reduction correlated with cortical thinning of the insula, primary sensory cortex/inferior parietal sulcus, and superior temporal polysensory areas. This indicates that TNFi treatment produces changes in brain areas implicated in sensory, motor, affective, and cognitive functions.

  1. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  2. Caspase-3, myogenic transcription factors and cell cycle inhibitors are regulated by leukemia inhibitory factor to mediate inhibition of myogenic differentiation

    PubMed Central

    2011-01-01

    Background Leukemia inhibitory factor (LIF) is known to inhibit myogenic differentiation as well as to inhibit apoptosis and caspase-3 activation in non-differentiating myoblasts. In addition caspase-3 activity is required for myogenic differentiation. Therefore the aim of this study was to further investigate mechanisms of the differentiation suppressing effect of LIF in particular the possibility of a caspase-3 mediated inhibition of differentiation. Results LIF dependent inhibition of differentiation appeared to involve several mechanisms. Differentiating myoblasts that were exposed to LIF displayed increased transcripts for c-fos. Transcripts for the cell cycle inhibitor p21 as well as muscle regulatory factors myoD and myogenin were decreased with LIF exposure. However, LIF did not directly induce a proliferative effect under differentiation conditions, but did prevent the proportion of myoblasts that were proliferating from decreasing as differentiation proceeded. LIF stimulation decreased the percentage of cells positive for active caspase-3 occurring during differentiation. Both the effect of LIF inhibiting caspase-3 activation and differentiation appeared dependent on mitogen activated protein kinase and extracellular signal regulated kinase kinase (MEK) signalling. The role of LIF in myogenic differentiation was further refined to demonstrate that myoblasts are unlikely to secrete LIF endogenously. Conclusions Altogether this study provides a more comprehensive view of the role of LIF in myogenic differentiation including LIF and receptor regulation in myoblasts and myotubes, mechanisms of inhibition of differentiation and the link between caspase-3 activation, apoptosis and myogenic differentiation. PMID:21798094

  3. Factors Associated with the Development of Drug Resistance Mutations in HIV-1 Infected Children Failing Protease Inhibitor-Based Antiretroviral Therapy in South Africa

    PubMed Central

    Melikian, George; van Dyk, Gisela; Thomas, Winifred; du Plessis, Nicolette M.; Avenant, Theunis

    2015-01-01

    Objective Limited data are available from the developing world on antiretroviral drug resistance in HIV-1 infected children failing protease inhibitor-based antiretroviral therapy, especially in the context of a high tuberculosis burden. We describe the proportion of children with drug resistance mutations after failed protease inhibitor-based antiretroviral therapy as well as associated factors. Methods Data from children initiated on protease inhibitor-based antiretroviral therapy with subsequent virological failure referred for genotypic drug resistance testing between 2008 and 2012 were retrospectively analysed. Frequencies of drug resistance mutations were determined and associations with these mutations identified through logistic regression analysis. Results The study included 65 young children (median age 16.8 months [IQR 7.8; 23.3]) with mostly advanced clinical disease (88.5% WHO stage 3 or 4 disease), severe malnutrition (median weight-for-age Z-score -2.4 [IQR -3.7;-1.5]; median height-for-age Z-score -3.1 [IQR -4.3;-2.4]), high baseline HIV viral load (median 6.04 log10, IQR 5.34;6.47) and frequent tuberculosis co-infection (66%) at antiretroviral therapy initiation. Major protease inhibitor mutations were found in 49% of children and associated with low weight-for-age and height-for-age (p = 0.039; p = 0.05); longer duration of protease inhibitor regimens and virological failure (p = 0.001; p = 0.005); unsuppressed HIV viral load at 12 months of antiretroviral therapy (p = 0.001); tuberculosis treatment at antiretroviral therapy initiation (p = 0.048) and use of ritonavir as single protease inhibitor (p = 0.038). On multivariate analysis, cumulative months on protease inhibitor regimens and use of ritonavir as single protease inhibitor remained significant (p = 0.008; p = 0.033). Conclusion Major protease inhibitor resistance mutations were common in this study of HIV-1-infected children, with the timing of tuberculosis treatment and subsequent

  4. In vitro and in vivo activities of novel, semisynthetic thiopeptide inhibitors of bacterial elongation factor Tu.

    PubMed

    Leeds, J A; LaMarche, M J; Brewer, J T; Bushell, S M; Deng, G; Dewhurst, J M; Dzink-Fox, J; Gangl, E; Jain, A; Lee, L; Lilly, M; Manni, K; Mullin, S; Neckermann, G; Osborne, C; Palestrant, D; Patane, M A; Raimondi, A; Ranjitkar, S; Rann, E M; Sachdeva, M; Shao, J; Tiamfook, S; Whitehead, L; Yu, D

    2011-11-01

    Recently, we identified aminothiazole derivatives of GE2270 A. These novel semisynthetic congeners, like GE2270 A, target the essential bacterial protein elongation factor Tu (EF-Tu). Medicinal chemistry optimization of lead molecules led to the identification of preclinical development candidates 1 and 2. These cycloalklycarboxylic acid derivatives show activity against difficult to treat Gram-positive pathogens and demonstrate increased aqueous solubility compared to GE2270 A. We describe here the in vitro and in vivo activities of compounds 1 and 2 compared to marketed antibiotics. Compounds 1 and 2 were potent against clinical isolates of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC(90) ≤ 0.25 μg/ml) but weaker against the streptococci (MIC(90) ≥ 4 μg/ml). Like GE2270 A, the derivatives inhibited bacterial protein synthesis and selected for spontaneous loss of susceptibility via mutations in the tuf gene, encoding EF-Tu. The mutants were not cross-resistant to other antibiotic classes. In a mouse systemic infection model, compounds 1 and 2 protected mice from lethal S. aureus infections with 50% effective doses (ED(50)) of 5.2 and 4.3 mg/kg, respectively. Similarly, compounds 1 and 2 protected mice from lethal systemic E. faecalis infections with ED(50) of 0.56 and 0.23 mg/kg, respectively. In summary, compounds 1 and 2 are active in vitro and in vivo activity against difficult-to-treat Gram-positive bacterial infections and represent a promising new class of antibacterials for use in human therapy.

  5. Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia-Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer

    DTIC Science & Technology

    2012-10-01

    respectively. Third , expression of HER2 and HIF-1 was also analyzed in another aromatase inhibitor (AI)-resistant cell line . Exemestane-resistant...combined with letrozole versus letrozole and placebo as first- line therapy for postmenopausal hormone receptor-positive metastatic breast cancer. J Clin...to identify other relevant factors involved that can be used as biomarkers of AI resistance or targets for therapy . One such factor may include HIF-1

  6. New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

    PubMed

    Alshaikh, N A; Rosing, J; Thomassen, M C L G D; Castoldi, E; Simioni, P; Hackeng, T M

    2017-02-17

    Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

  7. Abnormal production of tumor necrosis factor (TNF) -- alpha and clinical efficacy of the TNF inhibitor etanercept in a patient with PAPA syndrome [corrected].

    PubMed

    Cortis, Elisabetta; De Benedetti, Fabrizio; Insalaco, Antonella; Cioschi, Stefania; Muratori, Flaminia; D'Urbano, Leila E; Ugazio, Alberto G

    2004-12-01

    We report a family with pyogenic sterile arthritis, pyoderna and acne syndrome (PAPA). The proband presented several episodes of sterile pyogenic arthritis and became unresponsive to glucocorticoids. After treatment with the tumor necrosis factor inhibitor etanercept, the disease underwent rapid and sustained clinical remission. Production of tumor necrosis factor-alpha by mononuclear cells of the proband and of the affected relatives was abnormally elevated.

  8. Spitzer IRS Observations of the XA Region in the Cygnus Loop Supernova Remnant

    NASA Astrophysics Data System (ADS)

    Sankrit, Ravi; Raymond, John C.; Bautista, Manuel; Gaetz, Terrance J.; Williams, Brian J.; Blair, William P.; Borkowski, Kazimierz J.; Long, Knox S.

    2014-05-01

    We report on spectra of two positions in the XA region of the Cygnus Loop supernova remnant obtained with the InfraRed Spectrograph on the Spitzer Space Telescope. The spectra span the 10-35 μm wavelength range, which contains a number of collisionally excited forbidden lines. These data are supplemented by optical spectra obtained at the Whipple Observatory and an archival UV spectrum from the International Ultraviolet Explorer. Coverage from the UV through the IR provides tests of shock wave models and tight constraints on model parameters. Only lines from high ionization species are detected in the spectrum of a filament on the edge of the remnant. The filament traces a 180 km s-1 shock that has just begun to cool, and the oxygen to neon abundance ratio lies in the normal range found for Galactic H II regions. Lines from both high and low ionization species are detected in the spectrum of the cusp of a shock-cloud interaction, which lies within the remnant boundary. The spectrum of the cusp region is matched by a shock of about 150 km s-1 that has cooled and begun to recombine. The post-shock region has a swept-up column density of about 1.3 × 1018 cm-2, and the gas has reached a temperature of 7000-8000 K. The spectrum of the Cusp indicates that roughly half of the refractory silicon and iron atoms have been liberated from the grains. Dust emission is not detected at either position. Based on observations made with the Spitzer Space Telescope.

  9. Spitzer IRS observations of the XA region in the cygnus loop supernova remnant

    SciTech Connect

    Sankrit, Ravi; Bautista, Manuel; Williams, Brian J.; Blair, William P.; Borkowski, Kazimierz J.; Long, Knox S.

    2014-05-20

    We report on spectra of two positions in the XA region of the Cygnus Loop supernova remnant obtained with the InfraRed Spectrograph on the Spitzer Space Telescope. The spectra span the 10-35 μm wavelength range, which contains a number of collisionally excited forbidden lines. These data are supplemented by optical spectra obtained at the Whipple Observatory and an archival UV spectrum from the International Ultraviolet Explorer. Coverage from the UV through the IR provides tests of shock wave models and tight constraints on model parameters. Only lines from high ionization species are detected in the spectrum of a filament on the edge of the remnant. The filament traces a 180 km s{sup –1} shock that has just begun to cool, and the oxygen to neon abundance ratio lies in the normal range found for Galactic H II regions. Lines from both high and low ionization species are detected in the spectrum of the cusp of a shock-cloud interaction, which lies within the remnant boundary. The spectrum of the cusp region is matched by a shock of about 150 km s{sup –1} that has cooled and begun to recombine. The post-shock region has a swept-up column density of about 1.3 × 10{sup 18} cm{sup –2}, and the gas has reached a temperature of 7000-8000 K. The spectrum of the Cusp indicates that roughly half of the refractory silicon and iron atoms have been liberated from the grains. Dust emission is not detected at either position.

  10. Optimizing the use of epidermal growth factor receptor inhibitors in advanced non-small-lung cancer (NSCLC)

    PubMed Central

    Shash, Emad; Peccatori, Fedro Alessandro; Azim, Hatem A

    2011-01-01

    Lung cancer is the leading cause of cancer-related death in US and Europe. Treatment with a platinum-based chemotherapy remains the standard of care, however with modest effect on quality of life and overall survival which seldom reaches 1 year. Recently, several classes of targeted agents have emerged showing promising results. In particular, agents targeting the epidermal growth factor receptor (EGFR) showed impressive clinical activity both in the first line and salvage settings. However, it is evident that these drugs are not effective in all patients. Putting into consideration the very high cost of these agents, there is an urgent need to provide reliable tools to identify those patients that would derive the maximum benefit from these drugs. Several predictive biomarkers were developed to identify those patients who would derive the maximal benefit of these drugs. In this review we will discuss the recent updates on the role of EGFR inhibitors in the treatment of advanced NSCLC and the role of predictive bio-markers in patient selection. PMID:22263061

  11. Protein Inhibitor of Activated STAT Y (PIASy) Regulates Insulin Secretion by Interacting with LIM Homeodomain Transcription Factor Isl1

    PubMed Central

    Yan, Chengzhi; Yu, Chulin; Zhang, Di; Cui, Yan; Zhou, Jinlian; Cui, Sheng

    2016-01-01

    It is known that the LIM homeodomain transcription factor Isl1 is highly expressed in all pancreatic endocrine cells and functions in regulating pancreatic development and insulin secretion. The Isl1 mutation has been found to be associated with type 2 diabetes, but the mechanism responsible for Isl1 regulation of insulin synthesis and secretion still needs to be elucidated. In the present study, the protein inhibitor of activated STAT Y (PIASy) was identified as a novel Isl1-interacting protein with a yeast two-hybrid system, and its interaction with Isl1 was further confirmed by a co-immunoprecipitation experiment. PIASy and Isl1 colocalize in human and mouse pancreas and NIT beta cells. Furthermore, PIASy and Isl1 upregulate insulin gene expression and insulin secretion in a dose-dependent manner by activating the insulin promoter. PIASy and Isl1 mRNA expression levels were also increased in type 2 diabetic db/db mice. In addition, our results demonstrate that PIASy and Isl1 cooperate to activate the insulin promoter through the Isl1 homeodomain and PIASy ring domain. These data suggest that that PIASy regulates insulin synthesis and secretion by interacting with Isl1 and provide new insight into insulin regulation, although the detailed molecular mechanism needs to be clarified in future studies. PMID:28000708

  12. Three generations of epidermal growth factor receptor tyrosine kinase inhibitors developed to revolutionize the therapy of lung cancer

    PubMed Central

    Zhang, Haijun

    2016-01-01

    Lung cancer, ~80%–85% of which is non-small-cell lung cancer (NSCLC), is the leading cause of cancer-related mortality worldwide. Sensitizing mutations in epidermal growth factor receptor (EGFR) gene (EGFRm+), such as exon 19 deletions and exon 21 L858R point mutations, are the most important drivers in NSCLC patients. In this respect, small-molecule EGFR tyrosine kinase inhibitors (TKIs) have been designed and developed, which launched the era of targeted, personalized and precise medicine for lung cancer. Patients with EGFRm+ could achieve good responses to the treatment with the first-generation EGFR TKIs, such as erlotinib and gefitinib. However, most patients develop acquired drug resistance mostly driven by the T790M mutation occurring within exon 20. Although the second-generation EGFR TKIs, such as afatinib, dacomitinib and neratinib, demonstrated promising activity against T790M in preclinical models, they have failed to overcome resistance in patients due to dose-limiting toxicity. Recently, the third-generation EGFR TKIs have shown to be effective against cell lines and murine models harboring T790M mutations while sparing wild-type EGFR, which represents a promising breakthrough approach in overcoming T790M-mediated resistance in NSCLC patients. This article provides a comprehensive review of the therapy revolution for NSCLC with three generations of EGFR TKIs. PMID:27920501

  13. Cinnamide Derivatives of d‐Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa †

    PubMed Central

    Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Wagner, Stefanie; Audfray, Aymeric; Prestel, Andreas; Möller, Heiko M.; Imberty, Anne

    2015-01-01

    Abstract Pseudomonas aeruginosa is an opportunistic Gram‐negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate‐based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug‐like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d‐mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure–activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure‐based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half‐lives in the 5–20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti‐infectives against this multidrug‐resistant pathogen. PMID:27308201

  14. Combining oral contraceptives with a natural nuclear factor-kappa B inhibitor for the treatment of endometriosis-related pain

    PubMed Central

    Maia, Hugo; Haddad, Clarice; Casoy, Julio

    2014-01-01

    Endometriosis is a chronic disease in which a persistent state of heightened inflammation is maintained by nuclear factor-kappa B (NF-κB) activation. The progestins present in oral contraceptives are potent inhibitors of NF-κB translocation to cell nuclei, while Pycnogenol® (Pinus pinaster) acts by blocking post-translational events. In this study, the effects of Pycnogenol on pain scores were investigated in patients with endometriosis using oral contraceptives containing either gestodene or drospirenone in extended regimens. Pain scores were determined using a visual analog scale before and after 3 months of treatment. Oral contraceptives, used alone (groups 1 and 3) or in association with Pycnogenol (groups 2 and 4), resulted in significant decreases in pain scores after 3 months of treatment; however, this reduction was significantly greater in the groups using oral contraceptives + Pycnogenol (groups 2 and 4) compared with those using oral contraceptives alone (groups 1 and 3). In the groups using oral contraceptives alone, 50% of patients became pain-free by the end of the third month of treatment. These results suggest that Pycnogenol increases the efficacy of oral contraceptives for the treatment of endometriosis-related pain. PMID:24379702

  15. Ethacrynic acid improves the antitumor effects of irreversible epidermal growth factor receptor tyrosine kinase inhibitors in breast cancer

    PubMed Central

    Hu, YunLong; Chen, TingTing; Peng, BoYa; Gao, NingNing; Jin, ZhenChao; Jia, TieLiu; Zhang, Na; Wang, ZhuLin; Jin, GuangYi

    2016-01-01

    Prolonged treatment of breast cancer with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) often results in acquired resistance and a narrow therapeutic index. One strategy to improve the therapeutic effects of EGFR TKIs is to combine them with drugs used for other clinical indications. Ethacrynic acid (EA) is an FDA approved drug that may have antitumor effects and may enhance the cytotoxicity of chemotherapeutic agents by binding to glutathione and inhibiting WNT signaling. While the α,β-unsaturated-keto structure of EA is similar to that of irreversible TKIs, the mechanism of action of EA when combined with irreversible EGFR TKIs in breast cancer remains unknown. We therefore investigated the combination of irreversible EGFR TKIs and EA. We found that irreversible EGFR TKIs and EA synergistically inhibit breast cancer both in vitro and in vivo. The combination of EGFR TKIs and EA induces necrosis and cell cycle arrest and represses WNT/β-catenin signaling as well as MAPK-ERK1/2 signaling. We conclude that EA synergistically enhances the antitumor effects of irreversible EGFR TKIs in breast cancer. PMID:27487128

  16. Ethacrynic acid improves the antitumor effects of irreversible epidermal growth factor receptor tyrosine kinase inhibitors in breast cancer.

    PubMed

    Liu, Bing; Huang, XinPing; Hu, YunLong; Chen, TingTing; Peng, BoYa; Gao, NingNing; Jin, ZhenChao; Jia, TieLiu; Zhang, Na; Wang, ZhuLin; Jin, GuangYi

    2016-09-06

    Prolonged treatment of breast cancer with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) often results in acquired resistance and a narrow therapeutic index. One strategy to improve the therapeutic effects of EGFR TKIs is to combine them with drugs used for other clinical indications. Ethacrynic acid (EA) is an FDA approved drug that may have antitumor effects and may enhance the cytotoxicity of chemotherapeutic agents by binding to glutathione and inhibiting WNT signaling. While the α,β-unsaturated-keto structure of EA is similar to that of irreversible TKIs, the mechanism of action of EA when combined with irreversible EGFR TKIs in breast cancer remains unknown. We therefore investigated the combination of irreversible EGFR TKIs and EA. We found that irreversible EGFR TKIs and EA synergistically inhibit breast cancer both in vitro and in vivo. The combination of EGFR TKIs and EA induces necrosis and cell cycle arrest and represses WNT/β-catenin signaling as well as MAPK-ERK1/2 signaling. We conclude that EA synergistically enhances the antitumor effects of irreversible EGFR TKIs in breast cancer.

  17. Enhancing the secretory yields of leech carboxypeptidase inhibitor in Escherichia coli: influence of trigger factor and signal recognition particle.

    PubMed

    Puertas, Juan-Miguel; Nannenga, Brent L; Dornfeld, Kevin T; Betton, Jean-Michel; Baneyx, François

    2010-11-01

    The signal recognition particle (SRP) dependent secretion pathway is as an attractive alternative to Sec-dependent export for the production of disulfide-bonded and/or fast-folding recombinant proteins in the Escherichia coli periplasm. SRP, which shares a ribosomal attachment site with the molecular chaperone trigger factor (TF), recognizes highly hydrophobic signal sequence as they emerge from the ribosome and delivers ribosome nascent chain complexes to FtsY for subsequent cotranslational translocation of target proteins across the SecYEG pore. However, like in the case of Sec-dependent export, secretory yields can be limited by the accumulation of precursor proteins in the cytoplasm. Using leech carboxypeptidase inhibitor (LCI) fused to the SRP-dependent DsbA signal sequence as a model system, we show that a null mutation in the gene encoding TF (Deltatig) or SRP co-expression reduce pre-LCI accumulation by half, and that quantitative export can be achieved by combining the two strategies. Interestingly, enhanced precursor processing did not alter periplasmic LCI levels but increased the amount of protein excreted in the growth medium. All mature LCI was nearly fully active and an 80% increase in productivity was achieved in Deltatig cells alone due to their faster growth. Our results show that competition between SRP and TF can interfere with efficient export of recombinant proteins targeted to the SRP pathway and establish TF-deficient strains and SRP co-expression as a simple solution to improve yields.

  18. Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains.

    PubMed

    Gharagozlou, Soheila; Sharifian, Ramazan A; Khoshnoodi, Jalal; Karimi, Katayoon; Milani, Monica; Okita, David K; Shokri, Fazel; Conti-Fine, Bianca M

    2009-05-01

    Development of antibodies (Ab) that either block the function of coagulation factor VIII (FVIII) (inhibitors) or clear it from circulation, seriously complicate the treatment of haemophilia A patients with FVIII products. Autoantibodies which develop in subjects without congenital FVIII defects, cause acquired haemophilia, a rare but life-threatening coagulopathy. Identification of the FVIII epitopes to which inhibitor Abs bind will help understanding the mechanisms of inhibitor activity, and perhaps development of new therapies. Here, we examined the FVIII peptide sequence regions recognised by anti-FVIII Ab in the plasma of six congenital and one acquired haemophilia patients with high inhibitor titers (24.4-2000 BU/ml). We used indirect ELISA and overlapping synthetic peptides, 20 residues long, spanning the sequence of the A and C FVIII domains. None of the plasma samples reacted with A1, A3 or C1 domain peptides. Six plasmas reacted with A2 and/or C2 peptides. Peptides spanning residues A2-521-690 and C2-2251-2332 were recognised most frequently and strongly. They include residues that contribute to the binding sites for activated factor IX and phosphatidyl serine/von Willebrand factor. These results suggest that anti-FVIII Abs share a pattern of antigen specificity in our panel of patients, and that exposed regions of the FVIII molecule that form functionally important binding sites elicit an intense Ab response.

  19. An in silico approach for identification of novel inhibitors as a potential therapeutics targeting HIV-1 viral infectivity factor.

    PubMed

    Sinha, Chanda; Nischal, Anuradha; Bandaru, Srinivas; Kasera, Priyadarshani; Rajput, Ashish; Nayarisseri, Anuraj; Khattri, Sanjay

    2015-01-01

    Currently available antiviral drugs target the pol-encoded retroviral enzymes or integrases, in addition, inhibitors that target HIV-1 envelope-receptor interactions have also been recently approved. Recent understanding of the interactions between HIV-1 and host restriction factors has provided fresh avenues for development of novel antiviral drugs. For example, viral infectivity factor (Vif) now surfaced as an important therapeutic target in treatment of HIV infection. Vif suppresses A3G antiviral activity by targeting these proteins for polyubiquitination and proteasomal degradation. In the present study we analyzed the inhibitory potential of VEC5 and RN18 to inhibit the Vif-A3G interaction through protein- protein docking studies. Perusal of the study showed that, VEC5 and RN18 though inhibits the interaction however showed sub optimal potential. To overcome this set back, we identified 35 structural analogues of VEC5 and 18 analogues of RN18 through virtual screening approach. Analogue with PubCID 71624757 and 55358204 (AKOS006479723) -structurally akin to VEC5 and RN18 respectively showed much appreciable interaction than their respective parent compound. Evident from Vif-A3G; protein - protein docking studies, analogue PubCID 71624757 demonstrated 1.08 folds better inhibitory potential than its parent compound VEC5 while analogue PubCID 55358204 was 1.15 folds better than RN18. Further these analogues passed drug likeness filters and predicted to be non- toxic. We expect these analogues can be put to pharmacodynamic studies that can pave way the breakthrough in HIV therapeutics.

  20. The cannabinoid dexanabinol is an inhibitor of the nuclear factor-kappa B (NF-kappa B).

    PubMed

    Jüttler, Eric; Potrovita, Ioana; Tarabin, Victoria; Prinz, Simone; Dong-Si, Tuan; Fink, George; Schwaninger, Markus

    2004-09-01

    Exogenous and endogenous cannabinoids have been shown to have neuroprotective effects in vitro and in vivo. Although many of the pharmacological effects of cannabinoids have been identified, the mechanism of neuroprotection still represents a controversy. Here we demonstrate for the first time protective effects of the synthetic cannabinoid dexanabinol by inhibiting apoptosis in a neuron-like cell line using nuclear staining and FACS analysis and in primary neurons. We provide further evidence of inhibition of nuclear factor-kappakappa B (NF-kappaB) by dexanabinol: Dexanabinol inhibits (1) phosphorylation and degradation of the inhibitor of NF-kappaB IkappaBalpha and translocation of NF-kappaB to the nucleus; dexanabinol reduces (2) the transcriptional activity of NF-kappaB and (3) mRNA accumulation of the NF-kappaB target genes tumor necrosis factor-alpha and interleukin-6 (TNF-alpha and IL-6). Dexanabinol does not bind to cannabinoid (CB) receptors 1 and 2. To investigate the mechanism of action, we employed the non-antioxidant CB1 receptor agonist WIN 55,212-2 and the antioxidant cannabinol, which binds to CB1 receptors only weakly. Both cannabinoids mimicked the effect of dexanabinol on NF-kappaB and apoptosis. This suggests that neither the antioxidant properties of cannabinoids nor binding to CB1 or CB2 receptors are responsible for the inhibition of NF-kappaB activity and apoptosis. Our results clearly demonstrate that dexanabinol inhibits NF-kappaB. NF-kappaB has been shown to be involved in brain damage and to promote neuronal cell death in vitro and in in vivo models of ischemic and neurodegenerative neurological diseases.

  1. Efficacy of unfractionated heparin, low molecular weight heparin and both combined for releasing total and free tissue factor pathway inhibitor.

    PubMed

    Altman, R; Scazziota, A; Rouvier, J

    1998-01-01

    Unfractionated heparin (UFH) exerts its anticoagulant properties by increasing the inactivation of thrombin and activated factor X by antithrombin III. Apart from this main action release of tissue factor pathway inhibitor (TFPI) from endothelial cells could also be important for the antithrombotic activity of heparins. Four different heparin preparations were injected subcutaneously into 5 healthy volunteers 1 week apart: (1) UFH 2,500 IU fix dose (FixUFH), (2) 1 mg/kg body weight of low molecular weight heparin (LMWH), (3) the combined LMWH-adjusted dose plus UFH 2,500 IU fix dose (ComHep) and (4) UFH 2,500 IU/10 kg body weight (UFHvar). Plasma samples were drawn before and 1, 2, 4, 6, 12 and 24 h afterwards. FixUFH did not affect the concentration of total and free TFPI. Total TFPI increased in the 1st hour after LMWH injection from 74 to 124 ng/ml (p < 0.01), after ComHep from 82 to 144 ng/ml (p < 0.01), and after UFHvar from 91 to 113 ng/ml (p < 0.05). All observed elevations were significant at the peak value (+/- 2 h, p < 0.01 compared with baselines). The increase of free TFPI produced by UFHvar (74.5 and 70.5 ng/ml) was significantly higher than with LMWH (42.8 and 38.0 ng/ml) at 2 and 4 h (p < 0.001 and p < 0.01, respectively). UFHvar and ComHep but not LMWH produced a statistically significant increase of free TFPI compared with FixUFH at 2, 4 and 6 h (p < 0. 01). We concluded that at comparable therapeutic doses, subcutaneous UFHvar released more free TFPI than LMWH and ComHep. A synergism between LMWH and low dose of UFH was found in 4-, 6- and 12-hour blood samples.

  2. PK10453, a nonselective platelet-derived growth factor receptor inhibitor, prevents the progression of pulmonary arterial hypertension

    PubMed Central

    2014-01-01

    Abstract The platelet-derived growth factor (PDGF) signaling pathway has been found to be activated in human pulmonary arterial hypertension (PAH) and in animal models of the disease. Our study tested the hypothesis that a novel, nonselective inhaled PDGF receptor inhibitor, PK10453, would decrease pulmonary hypertension both in the rat monocrotaline (MCT) model and the rat MCT plus pneumonectomy (MCT+PN) model of PAH. PK10453, delivered by inhalation for 4 (D4)- and 8 (D8)-minute exposures 3 times a day for 2 weeks, decreased right ventricular systolic pressure (RVSP) in both the rat MCT and rat MCT+PN models: RVSP was 80.4 ± 2.6 mmHg in the vehicle MCT group (n = 6), 44.4 ± 5.8 mmHg in the D4 MCT group (n = 6), and 37.1 ± 4.5 mmHg in the D8 MCT group (n = 5; P < 0.001 vs. vehicle); RVSP was 75.7 ± 7.1 mmHg in the vehicle MCT+PN group (n = 9), 40.4 ± 2.7 mmHg in the D4 MCT+PN group (n = 10), and 43.0 ± 3.0 mmHg in the D8 MCT+PN group (n = 8; P < 0.001). In the rat MCT+PN model, continuous telemetry monitoring of pulmonary artery pressures also demonstrated that PK10453 prevented the progression of PAH. Imatinib given by inhalation was equally effective in the MCT model but was not effective in the MCT+PN model. Immunohistochemistry demonstrated increased activation of the PDGFβ receptor compared to the PDGFα receptor in neointimal and perivascular lesions found in the MCT+PN model. We show that imatinib is selective for the PDGFα receptor, whereas PK10453 has a lower half-maximal inhibitor concentration (IC50) for inhibition of kinase activity of both the PDGFα and PDGFβ receptors compared to imatinib. In conclusion, PK10453, when delivered by inhalation, significantly decreased the progression of PAH in the rat MCT and MCT+PN models. Nonselective inhibition of both the PDGFα and PDGFβ receptors may have a therapeutic advantage over selective PDGFα receptor inhibition in PAH. PMID:25006424

  3. Pyrido[2,3-d]pyrimidin-5-ones: A Novel Class of Antiinflammatory Macrophage Colony-Stimulating Factor-1 Receptor Inhibitors

    SciTech Connect

    Huang, Hui; Hutta, Daniel A.; Rinker, James M.; Hu, Huaping; Parsons, William H.; Schubert, Carsten; DesJarlais, Renee L.; Crysler, Carl S.; Chaikin, Margery A.; Donatelli, Robert R.; Chen, Yanmin; Cheng, Deping; Zhou, Zhao; Yurkow, Edward; Manthey, Carl L.; Player, Mark R.

    2010-10-01

    A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound 37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In this model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid arthritis.

  4. Development of epidermal growth factor receptor tyrosine kinase inhibitors against EGFR T790M. Mutation in non small-cell lung carcinoma

    PubMed Central

    Wang, Yuli; Guo, Zhitao; Li, Yang

    2016-01-01

    Abstract Individualized therapies targeting epidermal growth factor receptor (EGFR) mutations show promises for the treatment of non small-cell lung carcinoma (NSCLC). However, disease progression almost invariably occurs 1 year after tyrosine kinase inhibitor (TKI) treatment. The most prominent mechanism of acquired resistance involves the secondary EGFR mutation, namely EGFR T790M, which accounts for 50%–60% of resistant tumors. A large amount of studies have focused on the development of effective strategies to treat TKI-resistant EGFR T790M mutation in lung tumors. Novel generations of EGFR inhibitors are producing encouraging results in patients with acquired resistance against EGFR T790M mutation. This review will summarize the novel inhibitors, which might overcome resistance against EGFR T790M mutation. PMID:28352770

  5. Vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors: development and validation of predictive 3-D QSAR models through extensive ligand- and structure-based approaches

    NASA Astrophysics Data System (ADS)

    Ragno, Rino; Ballante, Flavio; Pirolli, Adele; Wickersham, Richard B.; Patsilinakos, Alexandros; Hesse, Stéphanie; Perspicace, Enrico; Kirsch, Gilbert

    2015-08-01

    Vascular endothelial growth factor receptor-2, (VEGFR-2), is a key element in angiogenesis, the process by which new blood vessels are formed, and is thus an important pharmaceutical target. Here, 3-D quantitative structure-activity relationship (3-D QSAR) were used to build a quantitative screening and pharmacophore model of the VEGFR-2 receptors for design of inhibitors with improved activities. Most of available experimental data information has been used as training set to derive optimized and fully cross-validated eight mono-probe and a multi-probe quantitative models. Notable is the use of 262 molecules, aligned following both structure-based and ligand-based protocols, as external test set confirming the 3-D QSAR models' predictive capability and their usefulness in design new VEGFR-2 inhibitors. From a survey on literature, this is the first generation of a wide-ranging computational medicinal chemistry application on VEGFR2 inhibitors.

  6. Design, Synthesis and Biological Evaluation of 6-(2,6-Dichloro-3,5-dimethoxyphenyl)-4-substituted-1H-indazoles as Potent Fibroblast Growth Factor Receptor Inhibitors.

    PubMed

    Zhang, Zhen; Zhao, Dongmei; Dai, Yang; Cheng, Maosheng; Geng, Meiyu; Shen, Jingkang; Ma, Yuchi; Ai, Jing; Xiong, Bing

    2016-10-23

    Tyrosine kinase fibroblast growth factor receptor (FGFR), which is aberrant in various cancer types, is a promising target for cancer therapy. Here we reported the design, synthesis, and biological evaluation of a new series of 6-(2,6-dichloro-3,5-dimethoxyphenyl)-4-substituted-1H-indazole derivatives as potent FGFR inhibitors. The compound 6-(2,6-dichloro-3,5-dimethoxyphenyl)-N-phenyl-1H-indazole-4-carboxamide (10a) was identified as a potent FGFR1 inhibitor, with good enzymatic inhibition. Further structure-based optimization revealed that 6-(2,6-dichloro-3,5-dimethoxyphenyl)-N-(3-(4-methylpiperazin-1-yl)phenyl)-1H-indazole-4-carboxamide (13a) is the most potent FGFR1 inhibitor in this series, with an enzyme inhibitory activity IC50 value of about 30.2 nM.

  7. The intron-22–inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics

    PubMed Central

    Lozier, Jay N.; Kasper, Carol K.; Yanover, Chen; Nichols, Timothy

    2015-01-01

    Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive “intracellular (I)-FVIII-CRM” status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire. PMID:25406352

  8. Heat shock protein 90 (HSP90) inhibitors activate the heat shock factor 1 (HSF1) stress response pathway and improve glucose regulation in diabetic mice.

    PubMed

    Lee, Jee-Hyung; Gao, Jiaping; Kosinski, Penelope A; Elliman, Stephen J; Hughes, Thomas E; Gromada, Jesper; Kemp, Daniel M

    2013-01-18

    The cytoprotective stress response factor HSF1 regulates the transcription of the chaperone HSP70, which exhibits anti-inflammatory effects and improves insulin sensitivity. We tested the therapeutic potential of this pathway in rodent models of diabetes using pharmacological tools. Activation of the HSF1 pathway was achieved using potent inhibitors of the upstream regulatory protein, HSP90. Treatment with AUY922, a selective HSP90 inhibitor led to robust inhibition of JNK1 phosphorylation, cytoprotection and improved insulin signaling in cells, consistent with effects observed with HSP70 treatment. Chronic dosing with HSP90 inhibitors reversed hyperglycemia in the diabetic db/db mouse model, and improved insulin sensitivity in the diet-induced obese mouse model of insulin resistance, further supporting the concept that the HSF1 pathway is a potentially viable anti-diabetes target.

  9. Tumour necrosis factor inhibitors versus combination intensive therapy with conventional disease modifying anti-rheumatic drugs in established rheumatoid arthritis: TACIT non-inferiority randomised controlled trial

    PubMed Central

    Ibrahim, Fowzia; Farewell, Vern; O’Keeffe, Aidan G; Walker, David; Kelly, Clive; Birrell, Fraser; Chakravarty, Kuntal; Maddison, Peter; Heslin, Margaret; Patel, Anita; Kingsley, Gabrielle H

    2015-01-01

    Objective To determine whether intensive combinations of synthetic disease modifying drugs can achieve similar clinical benefits at lower costs to high cost biologics such as tumour necrosis factor inhibitors in patients with active rheumatoid arthritis resistant to initial methotrexate and other synthetic disease modifying drugs. Design Open label pragmatic randomised multicentre two arm non-inferiority trial over 12 months. Setting 24 rheumatology clinics in England. Participants Patients with rheumatoid arthritis who were eligible for treatment with tumour necrosis factor inhibitors according to current English guidance were randomised to either the tumour necrosis factor inhibitor strategy or the combined disease modifying drug strategy. Interventions Biologic strategy: start tumour necrosis factor inhibitor; second biologic in six month for non-responders. Alternative strategy: start combination of disease modifying drugs; start tumour necrosis factor inhibitors after six months in non-responders. Main outcome measure Primary outcome: reduction in disability at 12 months measured with patient recorded heath assessment questionnaire (range 0.00-3.00) with a 0.22 non-inferiority margin for combination treatment versus the biologic strategy. Secondary outcomes: quality of life, joint damage, disease activity, adverse events, and costs. Intention to treat analysis used multiple imputation methods for missing data. Results 432 patients were screened: 107 were randomised to tumour necrosis factor inhibitors and 101 started taking; 107 were randomised to the combined drug strategy and 104 started taking the drugs. Initial assessments were similar; 16 patients were lost to follow-up (seven with the tumour necrosis factor inhibitor strategy, nine with the combined drug strategy); 42 discontinued the intervention but were followed-up (19 and 23, respectively). The primary outcome showed mean falls in scores on the health assessment questionnaire of −0.30 with the

  10. Urinary Tissue Inhibitor of Metalloproteinase-2 and Insulin-Like Growth Factor-Binding Protein 7 for Risk Stratification of Acute Kidney Injury in Patients With Sepsis

    PubMed Central

    Honore, Patrick M.; Nguyen, H. Bryant; Gong, Michelle; Chawla, Lakhmir S.; Bagshaw, Sean M.; Artigas, Antonio; Shi, Jing; Joannes-Boyau, Olivier; Vincent, Jean-Louis

    2016-01-01

    Objectives: To examine the performance of the urinary biomarker panel tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 in patients with sepsis at ICU admission. To investigate the effect of nonrenal organ dysfunction on tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 in this population. Method: In this ancillary analysis, we included patients with sepsis who were enrolled in either of two trials including 39 ICUs across Europe and North America. The primary endpoint was moderate-severe acute kidney injury (equivalent to Kidney Disease Improving Global Outcome stage 2–3) within 12 hours of enrollment. We assessed biomarker performance by calculating the area under the receiver operating characteristic curve, sensitivity, specificity, and negative and positive predictive values at three cutoffs: 0.3, 1.0, and 2.0 (ng/mL)2/1,000. We also calculated nonrenal Sequential Organ Failure Assessment scores for each patient on enrollment and compared tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 results in patients with and without acute kidney injury and across nonrenal Sequential Organ Failure Assessment scores. Finally, we constructed a clinical model for acute kidney injury in this population and compared the performance of the model with and without tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7. Results: We included 232 patients in the analysis and 40 (17%) developed acute kidney injury. We observed significantly higher urine tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 in patients with acute kidney injury than without acute kidney injury in both patients with low and high nonrenal Sequential Organ Failure Assessment scores (p < 0.001). The area under the receiver operating characteristic curve (95% CI) of tissue inhibitor of metalloproteinase-2 and insulin

  11. The Rates of Serious Infections in HIV-infected Patients Who Received Tumor Necrosis Factor (TNF)-α Inhibitor Therapy for Concomitant Autoimmune Diseases

    PubMed Central

    Wangsiricharoen, Sintawat; Ligon, Colin; Gedmintas, Lydia; Dehrab, Admad; Tungsiripat, Marisa; Bingham, Clifton; Lozada, Carlos; Calabrese, Leonard

    2016-01-01

    Objectives To estimate the incidence of serious infections in patients with HIV infection and autoimmune disease who were treated with tumor necrosis factor (TNF) -α inhibitor therapy, and to compare these rates among stratified viral load levels. Methods Using a unified search strategy, four centers identified HIV-infected patients exposed to TNF-α inhibitors. Patient characteristics and infection data were assessed via chart review in all patients who were ≥18 years old and received TNF-α inhibitor therapy after HIV diagnosis between January 1999 and March 2015. Results Twenty-three patients with 26 uses of TNF-α inhibitor therapy provided 86.7 person-years of follow-up. Two (8.7%) experienced at least 1 serious infection episode, an overall incidence rate of 2.55 per 100 patient-years (95% CI 0.28–9.23). The incidence rate per 100 patient-years was 3.28 (95% CI 0.04–18.26) among patients with viral load > 500 copies/mL at therapy initiation and 2.09 (0.03–11.65) among patients with viral load ≤ 500 copies/mL. Conclusion This study suggests that TNF-α inhibitors may have a comparable rate of serious infections to the range of those observed in registry databases when used in patients with HIV infection under active care. PMID:27332039

  12. Epidermal growth factor receptor double targeting by a tyrosine kinase inhibitor (Iressa) and a monoclonal antibody (Cetuximab). Impact on cell growth and molecular factors

    PubMed Central

    Fischel, J-L; Formento, P; Milano, G

    2005-01-01

    Among the recent advances in the molecular targeted therapy of cancer, the applications focused on epidermal growth factor receptor (EGFR) are currently the most promising and the most advanced at clinical level. In view of the different modes of action of monoclonal antibodies and tyrosine kinase inhibitors (TKI), it is tempting to examine the effect of a combination between these two EGFR targeting approaches. It was the purpose of the present study to test this combination at experimental level by using two epidermoid human cell lines CAL 33 and CAL 39. As C225 (Cetuximab®) and ZD1839 (Iressa®) are, respectively, the most clinically advanced drugs in the category of anti-EGFR drugs, the experiments were performed using these two representative compounds. The combination of C225 and ZD1839 was antagonistic whatever the cell line considered. These antagonistic effects were corroborated by molecular changes in apoptosis (PARP) and EGFR signalling (phospho-p42–44). Drugs alone led to a diminution in EGFR levels, while their combination increased the cellular expression in EGFR. These data suggest that new and tempting treatment strategies on the EGFR target consisting in a double hit with a monoclonal antibody and a TKI must be considered with caution. PMID:15756277

  13. Vandetanib (ZD6474), a dual inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinases: current status and future directions.

    PubMed

    Morabito, Alessandro; Piccirillo, Maria Carmela; Falasconi, Fabiano; De Feo, Gianfranco; Del Giudice, Antonia; Bryce, Jane; Di Maio, Massimo; De Maio, Ermelinda; Normanno, Nicola; Perrone, Francesco

    2009-04-01

    Vandetanib is a novel, orally available inhibitor of different intracellular signaling pathways involved in tumor growth, progression, and angiogenesis: vascular endothelial growth factor receptor-2, epidermal growth factor receptor, and REarranged during Transfection tyrosine kinase activity. Phase I clinical trials have shown that vandetanib is well tolerated as a single agent at daily doses < or =300 mg. In the phase II setting, negative results were observed with vandetanib in small cell lung cancer, metastatic breast cancer, and multiple myeloma. In contrast, three randomized phase II studies showed that vandetanib prolonged the progression-free survival (PFS) time of patients with non-small cell lung cancer (NSCLC) as a single agent when compared with gefitinib or when added to chemotherapy. Rash, diarrhea, hypertension, fatigue, and asymptomatic QTc prolongation were the most common adverse events. Antitumor activity was also observed in medullary thyroid cancer. Four randomized phase III clinical trials in NSCLC are exploring the efficacy of vandetanib in combination with docetaxel, the Zactima in cOmbination with Docetaxel In non-small cell lung Cancer (ZODIAC) trial, or with pemetrexed, the Zactima Efficacy with Alimta in Lung cancer (ZEAL) trial, or as a single agent, the Zactima Efficacy when Studied versus Tarceva (ZEST) and the Zactima Efficacy trial for NSCLC Patients with History of EGFR-TKI chemo-Resistance (ZEPHYR) trials. Based on a press release by the sponsor of these trials, the PFS time was longer with vandetanib in the ZODIAC and ZEAL trials; the ZEST trial was negative for its primary superiority analysis, but was successful according to a preplanned noninferiority analysis of PFS. Ongoing phase II and III clinical trials will better define the appropriate schedule, the optimal setting of evaluation, and the safety of long-term use of vandetanib.

  14. Protective Effect of Infliximab, a Tumor Necrosis Factor-Alfa Inhibitor, on Bleomycin-Induced Lung Fibrosis in Rats.

    PubMed

    Altintas, Nejat; Erboga, Mustafa; Aktas, Cevat; Bilir, Bulent; Aydin, Murat; Sengul, Aysun; Ates, Zehra; Topcu, Birol; Gurel, Ahmet

    2016-02-01

    We aimed to investigate the preventive effect of Infliximab (IFX), a tumor necrosis factor (TNF)-α inhibitor, on bleomycin (BLC)-induced lung fibrosis in rats. Rats were assigned into four groups as follows: I-BLC group, a single intra-tracheal BLC (2.5 mg/kg) was installed; II-control group, a single intra-tracheal saline was installed; III-IFX + BLC group, a single-dose IFX (7 mg/kg) was administered intraperitoneally (i.p.), 72 h before the intra-tracheal BLC installation; IV-IFX group, IFX (7 mg/kg) was administered alone i.p. on the same day with IFX + BLC group. All animals were sacrificed on the 14th day of BLC installation. Levels of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interleukin (IL)-6, periostin, YKL-40, nitric oxide (NO) in rat serum were measured, as well as, myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activity, and reduced glutathione (GSH), hydroxyproline, malondialdehyde (MDA) content in lung homogenates. Lung tissues were stained with hematoxylin and eosin (H&E) for quantitative histological evaluation. The inducible nitric oxide synthase (iNOS) expression and cell apoptosis in the lung tissues were determined quantitatively by immunohistochemical staining (INOS) and by TUNNEL staining, respectively. BLC installation worsened antioxidant status (such as SOD, CAT, GPx, GSH, MPO), while it increased the serum TNF-α, TGF-β, IL-6, periostin, YKL-40, and lipid peroxidation, and collagen deposition, measured by MDA and hydroxyproline, respectively. IFX pretreatment improved antioxidant status as well as BLC-induced lung pathological changes, while it decreased the TNF-α, TGF-β, IL-6, periostin, YKL-40, lipid peroxidation and collagen deposition. Finally, histological, immunohistochemical, and TUNNEL evidence also supported the ability of IFX to prevent BLC-induced lung fibrosis. The results of the present study indicate that IFX pretreatment can attenuate

  15. 18F-FDG PET/CT for Monitoring Treatment Responses to the Epidermal Growth Factor Receptor Inhibitor Erlotinib

    PubMed Central

    Walter, Franziska; Garon, Edward B.; Reckamp, Karen L.; Figlin, Robert; Phelps, Michael E.; Weber, Wolfgang A.; Czernin, Johannes; Allen-Auerbach, Martin S.

    2016-01-01

    Response rates of unselected non–small cell lung cancer (NSCLC) patients to the epidermal growth factor receptor inhibitor erlotinib are low and range from 10% to 20%. Early response assessments are needed to avoid costs and side effects of inefficient treatments. Here we determined whether early changes in tumor uptake of 18F-FDG can predict progression-free and overall survival in NSCLC patients who are treated with erlotinib. Methods Twenty-two patients (6 men, 16 women; mean age ± SD, 64 ± 13 y) with stage III or stage IV NSCLC who received erlotinib treatment were enrolled prospectively. 18F-FDG PET/CT was performed before the initiation of treatment (n = 22), after 2 wk (n = 22), and after 78 ± 21 d (n = 11). Tumor maximum standardized uptake values were measured for a maximum of 5 lesions for each patient. Tumor responses were classified using modified PET Response Criteria in Solid Tumors (use of maximum standardized uptake values). Median overall survival by Kaplan–Meier analysis was compared between groups using a log-rank test. Results The overall median time to progression was 52 d (95% confidence interval, 47–57 d). The overall median survival time was 131 d (95% confidence interval, 0–351 d). Patients with progressive metabolic disease on early follow-up PET showed a significantly shorter time to progression (47 vs. 119 d; P < 0.001) and overall survival (87 vs. 828 d; P = 0.01) than patients classified as having stable metabolic disease or partial or complete metabolic response. Conclusion These data suggest that 18F-FDG PET/CT performed early after the start of erlotinib treatment can help to identify patients who benefit from this targeted therapy. PMID:22045706

  16. The soluble guanylyl cyclase inhibitor NS-2028 reduces vascular endothelial growth factor-induced angiogenesis and permeability.

    PubMed

    Morbidelli, Lucia; Pyriochou, Anastasia; Filippi, Sandra; Vasileiadis, Ioannis; Roussos, Charis; Zhou, Zongmin; Loutrari, Heleni; Waltenberger, Johannes; Stössel, Anne; Giannis, Athanassios; Ziche, Marina; Papapetropoulos, Andreas

    2010-03-01

    Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses.

  17. Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice

    PubMed Central

    Verma, Dheeraj; Moghimi, Babak; LoDuca, Paul A.; Singh, Harminder D.; Hoffman, Brad E.; Herzog, Roland W.; Daniell, Henry

    2010-01-01

    To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin β-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20–25% of control animals survived after six to eight F.IX doses, 90–93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2–5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5–80 μg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (∼40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment. PMID:20351275

  18. Treatment effectiveness and treatment patterns among rheumatoid arthritis patients after switching from a tumor necrosis factor inhibitor to another medication

    PubMed Central

    Bonafede, Machaon MK; Curtis, Jeffrey R; McMorrow, Donna; Mahajan, Puneet; Chen, Chieh-I

    2016-01-01

    Objectives After treatment failure with a tumor necrosis factor inhibitor (TNFi), patients with rheumatoid arthritis (RA) can switch to another TNFi (TNFi cyclers) or to a targeted disease-modifying antirheumatic drug (DMARD) with a non-TNFi mechanism of action (non-TNFi switchers). This study compared treatment patterns and treatment effectiveness between TNFi cyclers and non-TNFi switchers in patients with RA. Methods The analysis included a cohort of patients from the Truven Health Analytics MarketScan Commercial database with RA who switched from a TNFi (adalimumab, certolizumab pegol, etanercept, golimumab, or infliximab) either to another TNFi or to a non-TNFi targeted DMARD (abatacept, tocilizumab, or tofacitinib) between January 1, 2010 and September 30, 2014. A claims-based algorithm was used to estimate treatment effectiveness based on six criteria (adherence, no dose increase, no new conventional therapy, no switch to another targeted DMARD, no new/increased oral glucocorticoid, and intra-articular injections on <2 days). Results The cohort included 5,020 TNFi cyclers and 1,925 non-TNFi switchers. Non-TNFi switchers were significantly less likely than TNFi cyclers to switch therapy again within 6 months (13.2% vs 19.5%; P<0.001) or within 12 months (29.7% vs 34.6%; P<0.001) and significantly more likely to be persistent on therapy at 12 months (61.8% vs 58.2%; P<0.001). Non-TNFi switchers were significantly more likely than TNFi cyclers to achieve all six of the claims-based effectiveness algorithm criteria for the 12 months after the initial switch (27% vs 24%; P=0.011). Conclusion Although the absolute differences were small, these results support switching to a non-TNFi targeted DMARD instead of TNFi cycling when patients with RA require another therapy after TNFi failure. PMID:27980429

  19. The effect of antenatal depression and selective serotonin reuptake inhibitor treatment on nerve growth factor signaling in human placenta.

    PubMed

    Kaihola, Helena; Olivier, Jocelien; Poromaa, Inger Sundström; Åkerud, Helena

    2015-01-01

    Depressive symptoms during pregnancy are common and may have impact on the developing child. Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed antidepressant treatment, but unfortunately, these treatments can also negatively affect the behavioral development and health of a child during pregnancy. In addition, serotonin (5-HT) exerts neurotrophic actions with thus far not fully known effects in the offspring. The neurotrophic growth factor (NGF) is involved in neuronal cell survival and differentiation, and altered placenta levels have been found to increase the risk for pregnancy complications, similar to those found in women treated with SSRIs. We therefore investigated whether the NGF signaling pathway was altered in the placenta from women treated with SSRIs (n = 12) and compared them with placenta from depressed (n = 12) and healthy mothers (n = 12). Results from immunohistochemical stainings revealed that placental NGF protein levels of SSRI-treated women were increased in both trophoblasts and endothelial cells compared with depressed and control women. In addition, downstream of the NGF receptor TrkA, increased levels of the signaling proteins ROCK2 and phosphorylated Raf-1 were found in stromal cells and a tendency towards increased levels of ROCK2 in trophoblasts and endothelial cells in SSRI-treated women when compared to healthy controls. SSRI-treated women also displayed increased levels of phosphorylated ROCK2 in all placental cell types studied in comparison with depressed and control women. Interestingly, in placental endothelial cells from depressed women, NGF levels were significantly lower compared to control women, but ROCK2 levels were increased compared with control and SSRI-treated women. Taken together, these results show that the NGF signaling and downstream pathways in the placenta are affected by SSRI treatment and/or antenatal depression. This might lead to an altered placental function, although the clinical

  20. Release of alpha 2-plasmin inhibitor from plasma fibrin clots by activated coagulation factor XIII. Its effect on fibrinolysis.

    PubMed Central

    Mimuro, J; Kimura, S; Aoki, N

    1986-01-01

    When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulation Factor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an acceleration of fibrinolysis which was proportional to the degree of the release of alpha 2PI. These results suggest the possibility that alpha 2PI could be released from fibrin in vivo by disrupting the equilibrium of the alpha 2PI-fibrin cross-linking reaction, and that the release would result in accelerated thrombolysis. Images PMID:2419360

  1. Sarilumab improves patient-reported outcomes in rheumatoid arthritis patients with inadequate response/intolerance to tumour necrosis factor inhibitors

    PubMed Central

    Strand, Vibeke; Reaney, Matthew; Chen, Chieh-I; Proudfoot, Clare W J; Guillonneau, Sophie; Bauer, Deborah; Mangan, Erin; Graham, Neil M H; van Hoogstraten, Hubert; Lin, Yong; Pacheco-Tena, César; Fleischmann, Roy

    2017-01-01

    Objective To evaluate effects of the anti-interleukin-6 receptor monoclonal antibody sarilumab administered with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) on patient-reported outcomes (PROs) in the TARGET trial in patients with rheumatoid arthritis (RA) with inadequate response or intolerance to tumour necrosis factor inhibitors (TNF-IR). Methods 546 patients (81.9% female, mean age 52.9 years) were randomised to placebo, sarilumab 150 or 200 mg subcutaneously every 2 weeks + csDMARDs. PROs included patient global assessment (PtGA); pain and morning stiffness visual analogue scales; Health Assessment Questionnaire Disability Index (HAQ-DI); Short Form-36 Health Survey (SF-36); FACIT-Fatigue (FACIT-F); Work Productivity Survey-Rheumatoid Arthritis (WPS-RA) and Rheumatoid Arthritis Impact of Disease (RAID). Changes from baseline at weeks 12 and 24 were analysed using a mixed model for repeated measures; post hoc analyses included percentages of patients reporting improvements ≥ minimum clinically important differences (MCID) and scores ≥ normative values. Results Sarilumab + csDMARDs doses resulted in improvements from baseline at week 12 vs placebo + csDMARDs in PtGA, pain, HAQ-DI, SF-36 and FACIT-F that were maintained at week 24. Sarilumab improved morning stiffness and reduced the impact of RA on work, family, social/leisure activities participation (WPS-RA) and on patients' lives (RAID). Percentages of patients reporting improvements ≥MCID and ≥ normative scores were greater with sarilumab than placebo. Conclusions In patients with TNF-IR RA, 150 and 200 mg sarilumab + csDMARDs resulted in clinically meaningful patient-reported benefits on pain, fatigue, function, participation and health status at 12 and 24 weeks that exceeded placebo + csDMARDs, and were consistent with the clinical profile previously reported. Trial registration number NCT01709578; Results. PMID:28326189

  2. Clinical Significance of Tissue Factor Pathway Inhibitor 2, a Serum Biomarker Candidate for Ovarian Clear Cell Carcinoma

    PubMed Central

    Arakawa, Noriaki; Kobayashi, Hiroshi; Yonemoto, Naohiro; Masuishi, Yusuke; Ino, Yoko; Shigetomi, Hiroshi; Furukawa, Naoto; Ohtake, Norihisa; Miyagi, Yohei; Hirahara, Fumiki; Hirano, Hisashi; Miyagi, Etsuko

    2016-01-01

    Background There is currently no reliable serum biomarker for ovarian clear cell carcinoma (CCC), a highly lethal histological subtype of epithelial ovarian cancer (EOC). Previously, using a proteome-based approach, we identified tissue factor pathway inhibitor 2 (TFPI2) as a candidate serum biomarker for CCC. In this study, we sought to evaluate the clinical diagnostic performance of TFPI2 in preoperative prediction of CCC. Methods Serum TFPI2 levels were measured in serum samples from a retrospective training set consisting of patients with benign and borderline ovarian tumors, EOC subtypes, and uterine diseases. Via receiver operating characteristic (ROC) analyses, we compared the diagnostic performance of TFPI2 with that of CA125 in discrimination of patients with ovarian CCC from other patient groups. The observed diagnostic performances were examined in a prospective validation set. Results The 268-patient training set included 29 patients with ovarian CCC. Unlike CA125, which was also elevated in patients with endometriosis and several EOC subtypes, serum TFPI2 levels were specifically elevated only in ovarian CCC patients, consistent with the mRNA expression pattern in tumor tissues. The area under the ROC curve (AUC) of serum TFPI2 was obviously higher than that of CA125 for discrimination of CCC from other ovarian diseases (AUC = 0.891 versus 0.595). Applying a cut-off value of 280 pg/mL, TFPI2 could distinguish early-stage (FIGO I and II) CCC from endometriosis with 72.2% sensitivity, 93.3% specificity, and 88.8% accuracy. Similar results were confirmed in an independent 156-patient prospective validation set. Conclusions TFPI2 is a useful serum biomarker for preoperative clinical diagnosis of CCC. PMID:27798689

  3. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer.

    PubMed

    Jackman, David; Pao, William; Riely, Gregory J; Engelman, Jeffrey A; Kris, Mark G; Jänne, Pasi A; Lynch, Thomas; Johnson, Bruce E; Miller, Vincent A

    2010-01-10

    Ten percent of North American patients with non-small-cell lung cancer have tumors with somatic mutations in the gene for the epidermal growth factor receptor (EGFR). Approximately 70% of patients whose lung cancers harbor somatic mutations in exons encoding the tyrosine kinase domain of EGFR experience significant tumor regressions when treated with the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. However, the overwhelming majority of these patients inevitably acquire resistance to either drug. Currently, the clinical definition of such secondary or acquired resistance is not clear. We propose the following criteria be used to define more precisely acquired resistance to EGFR TKIs. All patients should have the following criteria: previous treatment with a single-agent EGFR TKI (eg, gefitinib or erlotinib); either or both of the following: a tumor that harbors an EGFR mutation known to be associated with drug sensitivity or objective clinical benefit from treatment with an EGFR TKI; systemic progression of disease (Response Evaluation Criteria in Solid Tumors [RECIST] or WHO) while on continuous treatment with gefitinib or erlotinib within the last 30 days; and no intervening systemic therapy between cessation of gefitinib or erlotinib and initiation of new therapy. The relatively simple definition proposed here will lead to a more uniform approach to investigating the problem of acquired resistance to EGFR TKIs in this unique patient population. These guidelines should minimize reporting of false-positive and false-negative activity in these clinical trials and would facilitate the identification of agents that truly overcome acquired resistance to gefitinib and erlotinib.

  4. Safety of Resuming Tumor Necrosis Factor Inhibitors in Ankylosing Spondylitis Patients Concomitant with the Treatment of Active Tuberculosis: A Retrospective Nationwide Registry of the Korean Society of Spondyloarthritis Research

    PubMed Central

    Kim, Hye Won; Kwon, Seong Ryul; Jung, Kyong-Hee; Kim, Seong-Kyu; Baek, Han Joo; Seo, Mi Ryung; Bang, So-Young; Lee, Hye-Soon; Suh, Chang-Hee; Jung, Ju Yang; Son, Chang-Nam; Shim, Seung Cheol; Lee, Sang-Hoon; Lee, Seung-Geun; Lee, Yeon-Ah; Lee, Eun Young; Kim, Tae-Hwan

    2016-01-01

    Backgrounds Patients who develop an active tuberculosis infection during tumor necrosis factor (TNF) inhibitor treatment typically discontinue TNF inhibitor and receive standard anti-tuberculosis treatment. However, there is currently insufficient information on patient outcomes following resumption of TNF inhibitor treatment during ongoing anti- tuberculosis treatment. Our study was designed to investigate the safety of resuming TNF inhibitors in ankylosing spondylitis (AS) patients who developed tuberculosis as a complication of the use of TNF inhibitors. Methods Through the nationwide registry of the Korean Society of Spondyloarthritis Research, 3929 AS patients who were prescribed TNF inhibitors were recruited between June 2003 and June 2014 at fourteen referral hospitals. Clinical information was analyzed about the patients who experienced tuberculosis after exposure to TNF inhibitors. The clinical features of resumers and non-resumers of TNF inhibitors were compared and the outcomes of tuberculosis were surveyed individually. Findings Fifty-six AS patients were treated for tuberculosis associated with TNF inhibitors. Among them, 23 patients resumed TNF inhibitors, and these patients were found to be exposed to TNF inhibitors for a longer period of time and experienced more frequent disease flare-up after discontinuation of TNF inhibitors compared with those who did not resume. Fifteen patients resumed TNF inhibitors during anti-tuberculosis treatment (early resumers) and 8 after completion of anti-tuberculosis treatment (late resumers). Median time to resuming TNF inhibitor from tuberculosis was 3.3 and 9.0 months in the early and late resumers, respectively. Tuberculosis was treated successfully in all resumers and did not relapse in any of them during follow-up (median 33.8 [IQR; 20.8–66.7] months). Conclusions Instances of tuberculosis were treated successfully in our AS patients, even when given concomitantly with TNF inhibitors. We suggest that early

  5. Nuclear Factor kB and Inhibitor of kB: Acupuncture Protection Against Acute Focal Cerebral Ischemia in Rodents.

    PubMed

    Huang, Wei; Zhou, Zhongyu; Wan, Bijiang; Chen, Guang; Li, Jia

    2017-02-27

    Context • Acute, focal, cerebral ischemic stroke is a leading cause of morbidity and mortality worldwide. Acupuncture is an emerging alternative therapy for treatment of acute brain ischemia. Nevertheless, the precise mechanism underlying the neuroprotective effects of acupuncture has not been elucidated. Nuclear factor κB (NF-κB) and nuclear factor of κ light polypeptide gene enhancer in B cell inhibitor alpha (IκB-α) are involved in cerebral inflammation. However, the involvement of NF-κB and IκB-α in the protective effects of acupuncture on ischemic tolerance remains unknown. Objective • The study evaluated the hypothesis that acupuncture can exert a neuroprotective action in a rat model of middle cerebral artery occlusion (MCAO). Design • The rats were randomly divided into a normal group (N), a sham model group (SM), an MCAO model group (M), a sham acupuncture group (SA), and an acupuncture group (A). Setting • All of processes of this study were conducted at Hubei University of Chinese Medicine (Hubei Shang, China). Animals • The animals were 100 Sprague-Dawley rats, aged 3 mo. Intervention • Craniotomy and electrocoagulation of the middle cerebral artery were conducted to generate acute, focal, cerebral ischemic models in 3 groups, excluding the N and SM groups. The SM group received a surgical fenestration similar to the M group, but the procedure did not include the coagulation of the exposed artery. In the A group, acupuncture was administered at the acupoints Baihui (GV-20) and Renzhong (GV-26). In the SA group, sham acupuncture was performed at a depth of 5 mm at a position close to the left side of the GV-20 and GV-26 points. The N, M, and SM groups received neither the acupuncture nor the sham acupuncture treatment. Outcome Measures • The study (1) evaluated neurological function using the modified neurological severity score; (2) examined the ultrastructure; (3) assessed the infarct volume; (4) determined levels of serum

  6. Interactions outside the proteinase-binding loop contribute significantly to the inhibition of activated coagulation factor XII by its canonical inhibitor from corn.

    PubMed

    Korneeva, Vera A; Trubetskov, Mikhail M; Korshunova, Alena V; Lushchekina, Sofya V; Kolyadko, Vladimir N; Sergienko, Olga V; Lunin, Vladimir G; Panteleev, Mikhail A; Ataullakhanov, Fazoil I

    2014-05-16

    Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.

  7. Clinicopathological spectrum of kidney diseases in cancer patients treated with vascular endothelial growth factor inhibitors: a report of 5 cases and review of literature.

    PubMed

    Usui, Joichi; Glezerman, Ilya G; Salvatore, Steven P; Chandran, Chandra B; Flombaum, Carlos D; Seshan, Surya V

    2014-09-01

    Recently, cancer therapies have been supplemented by vascular endothelial growth factor (VEGF) inhibitors as anti-angiogenic agents. However, kidney-related adverse reactions associated with these agents clinically manifest as hypertension and proteinuria, the most severe form being thrombotic microangiopathy (TMA). We present the spectrum of pathological features in VEGF inhibitor-associated kidney disease. Clinicopathological findings of kidney disease were retrospectively studied in 5 cancer patients treated with anti-VEGF agents. Although 4 cases received bevacizumab (anti-VEGF-A), one was given sorafenib (small molecule tyrosine kinase inhibitor affecting VEGF-R2). All patients presented with acute kidney injury, hypertension, and/or proteinuria. All kidney biopsies showed recent and chronic endothelial injury of varying severity and vascular sclerosis, including 2 with typical active features of TMA. Furthermore, acute tubular injury with focal necrosis was seen in all cases. While administration of VEGF inhibitor was discontinued in 4 cases, it was resumed for 5 more doses, following steroid therapy in 1 case. Cessation of VEGF inhibitor therapy was successful in reversing anemia and led to improvement of hypertension and proteinuria in 4 of the 5 cases. One case with TMA progressed to end-stage renal disease. A range of renal pathologic lesions secondary to endothelial injury are noted often accompanied by acute tubular damage following anti-VEGF therapy, the most severe being TMA. While most of the clinical manifestations are reversible with discontinuation of therapy, the role of other nephrotoxic chemotherapeutic agents in enhancing renal injury including severe TMA and other host factors with possible poor outcome should be considered.

  8. Effect of 3.2 vs. 3.8% sodium citrate concentration on anti-Xa levels for patients on therapeutic low molecular weight heparin.

    PubMed

    Payne, S; MacKinnon, K; Keeney, M; Morrow, B; Kovacs, M J

    2003-10-01

    In this study, we compared the effect of sodium citrate, a sample collection variable, on the anti-Xa levels of patients (n = 28) on dalteparin, a low molecular weight heparin. The median anti-Xa level for 3.2% sodium citrate was 0.235 U/ml while the median level for 3.8% sodium citrate was 0.230 U/ml. We conclude that different sodium citrate concentrations give statistically equivalent anti-Xa levels for the same samples. This conclusion is in contrast to the findings of the effect of sodium citrate concentration on International Normalized Ratio (INR) and activated partial-thromboplastin time (aPTT). In accordance with previous recommendations, we advocate the exclusive use of 3.2% sodium citrate in an effort to standardize coagulation testing.

  9. AvrXa27 binding influences unwinding of the double-stranded DNA in the UPT box.

    PubMed

    Zhao, Jing; Zhang, Bo; Jiang, Junpeng; Liu, Nanv; Wei, Qi; Xi, Xuguang; Fu, Jing

    2017-03-04

    Transcription-Activator Like (TAL) effectors, delivered by Xanthomonas pathogens bind specifically to UP-regulated by TAL effectors (UPT) box of the host gene promoter to arouse disease or trigger defense response. This type of protein-DNA interaction model has been applied in site-directed genome editing. However, the off-target effects of TAL have severely hindered the development of this promising technology. To better exploit the specific interaction and to deeper understand the TAL-induced host transcription rewiring, the binding between the central repeat region (CRR) of the TAL effector AvrXa27 and its UPT box variants was studied by kinetics analysis and TAL-blocked helicase unwinding assay. The results revealed that while AvrXa27 exhibited the highest affinity to the wild type UPT box, it could also bind to mutated UPT box variants, implying the possibility of non-specific interactions. Furthermore, some of these non-specific combinations restricted the helicase-elicited double-stranded DNA (dsDNA) separation to a greater extent. Our findings provide insight into the mechanism of TAL transcriptional activation and are beneficial to TAL-mediated genome modification.

  10. Rice xa13 recessive resistance to bacterial blight is defeated by induction of the disease susceptibility gene Os-11N3.

    PubMed

    Antony, Ginny; Zhou, Junhui; Huang, Sheng; Li, Ting; Liu, Bo; White, Frank; Yang, Bing

    2010-11-01

    The rice (Oryza sativa) gene xa13 is a recessive resistance allele of Os-8N3, a member of the NODULIN3 (N3) gene family, located on rice chromosome 8. Os-8N3 is a susceptibility (S) gene for Xanthomonas oryzae pv oryzae, the causal agent of bacterial blight, and the recessive allele is defeated by strains of the pathogen producing any one of the type III effectors AvrXa7, PthXo2, or PthXo3, which are all members of the transcription activator-like (TAL) effector family. Both AvrXa7 and PthXo3 induce the expression of a second member of the N3 gene family, here named Os-11N3. Insertional mutagenesis or RNA-mediated silencing of Os-11N3 resulted in plants with loss of susceptibility specifically to strains of X. oryzae pv oryzae dependent on AvrXa7 or PthXo3 for virulence. We further show that AvrXa7 drives expression of Os-11N3 and that AvrXa7 interacts and binds specifically to an effector binding element within the Os-11N3 promoter, lending support to the predictive models for TAL effector binding specificity. The result indicates that variations in the TAL effector repetitive domains are driven by selection to overcome both dominant and recessive forms of resistance to bacterial blight in rice. The finding that Os-8N3 and Os-11N3 encode closely related proteins also provides evidence that N3 proteins have a specific function in facilitating bacterial blight disease.

  11. The fibroblast growth factor receptor genetic status as a potential predictor of the sensitivity to CH5183284/Debio 1347, a novel selective FGFR inhibitor.

    PubMed

    Nakanishi, Yoshito; Akiyama, Nukinori; Tsukaguchi, Toshiyuki; Fujii, Toshihiko; Sakata, Kiyoaki; Sase, Hitoshi; Isobe, Takehito; Morikami, Kenji; Shindoh, Hidetoshi; Mio, Toshiyuki; Ebiike, Hirosato; Taka, Naoki; Aoki, Yuko; Ishii, Nobuya

    2014-11-01

    The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F-driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations.

  12. Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid beta-protein precursor.

    PubMed

    Van Nostrand, W E; Schmaier, A H; Siegel, R S; Wagner, S L; Raschke, W C

    1995-09-29

    The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.

  13. The tissue factor pathway inhibitor 1 of Sciaenops ocellatus possesses antimicrobial activity and is involved in the immune response against bacterial infection.

    PubMed

    Zhang, Min; Sun, Li

    2011-03-01

    Tissue factor pathway inhibitor 1 (TFPI-1) is a Kunitz-type serine protease inhibitor that regulates the activation of tissue factor-induced coagulation. In teleosts, TFPI-1-like sequences have been found to exist in two species (Danio rerio and Cyprinus carpio); however, the potential function of fish TFPI-1 has not been investigated. In this study, we identified and analyzed a TFPI-1 homologue, SoTFPI-1, from red drum (Sciaenops ocellatus). The deduced amino acid sequence of SoTFPI-1 is 284 residues in length and contains three Kunitz domains, an acidic N-terminus, and a basic C-terminus. SoTFPI-1 shares 49.5% and 46.9% overall sequence identities with the TFPI-1 of D. rerio and C. carpio, respectively. Quantitative real time RT-PCR analysis showed that constitutive SoTFPI-1 expression occurred, in increasing order, in kidney, brain, liver, gill, blood, spleen, muscle, and heart. Bacterial infection and lipopolysaccharide exposure upregulated SoTFPI-1 expression in kidney in time-dependent manners. Recombinant SoTFPI-1 (rSoTFPI-1) purified from Escherichia coli exhibits not only serine protease inhibitor activity but also bactericidal activity in a manner that is independent of any host factors. A synthetic peptide, TO17, corresponding to the C-terminal basic region of SoTFPI-1 also possesses antibacterial effect that is more potent than that of the full-length rSoTFPI-1. Taken together, these results demonstrate that (i) SoTFPI-1 is a biologically active serine protease inhibitor endowed with bactericidal property; (ii) provide the first indication that teleost TFPI-1 is likely to be involved in anti-microbial infection and thus is linked to innate immune defense.

  14. Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1B

    SciTech Connect

    Shia, Steven; Stamos, Jennifer; Kirchhofer, Daniel; Fan, Bin; Wu, Judy; Corpuz, Raquel T.; Santell, Lydia; Lazarus, Robert A.; Eigenbrot, Charles

    2010-07-20

    Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 {angstrom} resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 {angstrom} resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

  15. Orchard factors associated with resistance and cross resistance to sterol demethylation inhibitor fungicides in populations of Venturia inaequalis from Pennsylvania.

    PubMed

    Pfeufer, Emily E; Ngugi, Henry K

    2012-03-01

    Orchard management practices, such as destroying of overwintered inoculum and limiting the number of fungicide applications, are often recommended as tactics for slowing the development of resistance to sterol demethylation-inhibitor (DMI) fungicides in populations of Venturia inaequalis. However, there is little quantitative evidence relating the use of such practices to levels of resistance in orchards. The aim of this study was to evaluate the sensitivity of V. inaequalis isolates from Pennsylvania to DMI fungicides, and to identify orchard management factors related to the incidence of resistant isolates. In total, 644 single-spore V. inaequalis cultures obtained from 20 apple orchards in 2008 or 2009 were tested for sensitivity to myclobutanil, fenbuconazole, or difenoconazole. Growers provided management history of the sampled plots. Widespread shifts toward resistance to the three fungicides were noted, with mean effective concentration for 50% inhibition (EC(50)) values of 2.136, 0.786, and 0.187 μg/ml for myclobutanil, fenbuconazole, and difenoconazole, respectively. Cross resistance to the three fungicides was documented in high correlation (Spearman's r > 0.6) between mean EC(50) values for 14 orchards. Based on a 0.5-μg/ml threshold, 66 and 26% of isolates were resistant to myclobutanil and fenbuconazole, respectively, and 22% were cross resistant to the two fungicides. A significant between-year shift toward increased resistance was noted in two of three orchards surveyed in both years. Failure to use dormant copper sprays, older trees, larger orchards, orchards with ≤10 cultivars, and application of >4 DMI sprays were positively correlated (0.0001 < P < 0.05) with the incidence of resistant isolates. Isolates from orchards with >4 DMI sprays were four times as likely to be resistant to fenbuconazole (odds ratio = 4.57; P = 0.015). Isolates from orchards without dormant copper sprays were twice as likely to be cross-shifted toward resistance to all

  16. Small Molecular Inhibitor of Transforming Growth Factor-{beta} Protects Against Development of Radiation-Induced Lung Injury

    SciTech Connect

    Anscher, Mitchell S. Thrasher, Bradley; Zgonjanin, Larisa; Rabbani, Zahid N.; Corbley, Michael J.; Fu Kai; Sun Lihong; Lee, W.-C.; Ling, Leona E.; Vujaskovic, Zeljko

    2008-07-01

    Purpose: To determine whether an anti-transforming growth factor-{beta} (TGF-{beta}) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. Methods and Materials: One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7, RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-{beta} were assessed. Results: The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-{beta}1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7g/kg nor for the short-course (3 weeks) SM16 at either dose level. Conclusion: SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-{beta} at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.

  17. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics.

    PubMed

    Chung, Clement

    2016-06-01

    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  18. Characterization and Pharmacological Properties of a Novel Multifunctional Kunitz Inhibitor from Erythrina velutina Seeds

    PubMed Central

    Machado, Richele J. A.; Monteiro, Norberto K. V.; Migliolo, Ludovico; Silva, Osmar N.; Pinto, Michele F. S.; Oliveira, Adeliana S.; Franco, Octávio L.; Kiyota, Sumika; Bemquerer, Marcelo P.; Uchoa, Adriana F.; Morais, Ana H. A.; Santos, Elizeu A.

    2013-01-01

    Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30–60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10−8 mol.L−1 and constant inhibition (Ki) of 1.0×10−8 mol.L−1, by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation. PMID

  19. Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

    PubMed

    Machado, Richele J A; Monteiro, Norberto K V; Migliolo, Ludovico; Silva, Osmar N; Pinto, Michele F S; Oliveira, Adeliana S; Franco, Octávio L; Kiyota, Sumika; Bemquerer, Marcelo P; Uchoa, Adriana F; Morais, Ana H A; Santos, Elizeu A

    2013-01-01

    Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.

  20. Elective surgery on factor VIII inhibitor patients using continuous infusion of recombinant activated factor VII: plasma factor VII activity of 10 IU/ml is associated with an increased incidence of bleeding.

    PubMed

    Smith, M P; Ludlam, C A; Collins, P W; Hay, C R; Wilde, J T; Grigeri, A; Melsen, T; Savidge, G F

    2001-10-01

    We examined recombinant activated factor VII (rVIIa) administered by continuous infusion to eight patients with inhibitors to factor VIII, undergoing elective surgery. rVIIa was infused at a fixed rate of 16.5 microg/kg/h for a median of 13.5 days (range 1-26). There was effective haemostasis at this infusion rate in only one of two minor procedures and two of six major operations. Three patients experienced excessive bleeding despite plasma factor VII activity around 10 IU/ml. Serious bleeding occurred in two other patients caused by procedural errors unrelated to rVIIa and required re-operation. The median rVIIa clearance on day 1 was 57 ml/h/kg (range 18-100) and on day 3 was 100 ml/h/kg (range 61-200). Clearance on the final infusion day was not significantly different from day 3. The infusion did not induce pathological activation of the coagulation mechanism. The only thrombotic adverse events were two episodes of superficial thrombophlebitis of the infused vein in one subject. In conclusion, the 16.5 microg/kg/h infusion rate reliably achieves plasma factor VII activity levels of 10 IU/ml, but this level does not provide reliable haemostasis.

  1. Targeting Vascular Endothelial Growth Factor Receptor 2 and Protein Kinase D1 Related Pathways by a Multiple Kinase Inhibitor in Angiogenesis and Inflammation Related Processes In Vitro

    PubMed Central

    Varga, Attila; Gyulavári, Pál; Greff, Zoltán; Futosi, Krisztina; Németh, Tamás; Simon-Szabó, Laura; Kerekes, Krisztina; Szántai-Kis, Csaba; Brauswetter, Diána; Kokas, Márton; Borbély, Gábor; Erdei, Anna; Mócsai, Attila; Kéri, György; Vántus, Tibor

    2015-01-01

    Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways. PMID:25874616

  2. Genetic polymorphisms of tumour necrosis factor alpha (TNF-α) promoter gene and response to TNF-α inhibitors in Spanish patients with inflammatory bowel disease.

    PubMed

    López-Hernández, R; Valdés, M; Campillo, J A; Martínez-Garcia, P; Salama, H; Salgado, G; Boix, F; Moya-Quiles, M R; Minguela, A; Sánchez-Torres, A; Miras, M; Garcia, A; Carballo, F; Álvarez-López, M R; Muro, M

    2014-02-01

    Tumour necrosis factor alpha (TNF-α) has an important role in inflammatory response. Alterations in the regulation of TNF-α have been implicated in a variety of inflammatory disorders, including Inflammatory bowel disease (IBD). Indeed, a common treatment for IBD is the use of TNF-α inhibitors. Polymorphisms in the TNF-α promoter region are known to affect the level of gene expression. Our aim was to investigate the influence of these single nucleotide polymorphisms (SNPs) in TNF-α promoter gene play in the risk of IBD in a Spanish population and their individual response to anti-TNF-α treatment. DNA samples from patients with IBD and controls were screened for TNF-α -238G/A (rs361525) and -308G/A (rs1800629) SNPs by PCR-SSOP using a microbeads luminex assay and compared with response to TNF-α inhibitors. There were not statistical differences in -238G/A and -308G/A allele and genotype frequencies between patients. However, we found an increased frequency of -308A allele and -308GA genotype in these nonresponders patients to TNF-α inhibitors with respect to responders patients (Pc < 0.05). This -308GA genotype has been classified as high producer of this cytokine. This fact could actually be interesting to explain the different response of patients with IBD with respect to TNF-α inhibitors. TNF-α promoter gene polymorphism does not seem to play a role in IBD susceptibility, but particular TNF-α genotypes may be involved in the different responses to TNF-α inhibitor treatment in Spanish patients with IBD.

  3. Targeting vascular endothelial growth factor receptor 2 and protein kinase D1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

    PubMed

    Varga, Attila; Gyulavári, Pál; Greff, Zoltán; Futosi, Krisztina; Németh, Tamás; Simon-Szabó, Laura; Kerekes, Krisztina; Szántai-Kis, Csaba; Brauswetter, Diána; Kokas, Márton; Borbély, Gábor; Erdei, Anna; Mócsai, Attila; Kéri, György; Vántus, Tibor

    2015-01-01

    Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways.

  4. PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1): A KEY FACTOR LINKING FIBRINOLYSIS AND AGE-RELATED SUBCLINICAL AND CLINICAL CONDITIONS

    PubMed Central

    Cesari, Matteo; Pahor, Marco; Incalzi, Raffaele Antonelli

    2010-01-01

    The close relationship existing between aging and thrombosis has growingly been studied in this last decade. The age-related development of a pro-thrombotic imbalance in the fibrinolysis homeostasis has been hypothesized at the basis of this increased cardiovascular and cerebrovascular risk. Fibrinolysis is the resulting of the interactions among multiple plasminogen activators and inhibitors constituing the enzymatic cascade, and ultimately leading to the degradation of fibrin. The plasminogen activator system plays a key role in a wide range of physiological and pathological processes. Plasminogen activator inhibitor-1 (PAI-1) is a member of the superfamily of serine-protease inhibitors (or serpins), and the principal inhibitor of both the tissue-type and the urinary-type plasminogen activator, the two plasminogen activators able to activate plasminogen. In this review, current evidence describing the central role played by PAI-1 in a number of age-related subclinical (i.e., inflammation, atherosclerosis, insulin resistance) and clinical (i.e., obesity, comorbidities, Werner syndrome) conditions is presented. Despite some controversial and unclear issues, PAI-1 represents an extremely promising marker which may become a biological parameter to be growingly considered in the prognostic evaluation, in the disease monitoring, and as treatment target of age-related conditions in the next future. PMID:20626406

  5. Case Report of Lichen Planopilaris Occurring in a Pediatric Patient Receiving a Tumor Necrosis Factor α Inhibitor and a Review of the Literature.

    PubMed

    Jayasekera, Prativa S A; Walsh, Maeve L; Hurrell, Daniel; Parslew, Richard A G

    2016-01-01

    A 12-year-old girl with extended oligoarthritis treated with adalimumab presented with a short history of a progressive cutaneous eruption involving the legs and scalp. Physical examination and histologic results were consistent with lichen planopilaris. The adalimumab was discontinued. She received treatment with topical clobetasol propionate and the majority of the lesions resolved. Residual lesions and the extended oligoarthritis were then treated with sulfasalazine. Adalimumab is a tumor necrosis factor α (TNF-α) inhibitor used for the treatment of a variety of immunologically mediated conditions, including lichen planus and lichen planopilaris. TNF-α antagonists have been associated with paradoxical psoriasiform, lichenoid, eczematous, granulomatous, and acneiform eruptions. We detail this case and review the literature of lichenoid reactions secondary to TNF-α inhibitors.

  6. Pyrazolo-pyrimidine-derived c-Src inhibitor reduces angiogenesis and survival of squamous carcinoma cells by suppressing vascular endothelial growth factor production and signaling.

    PubMed

    Donnini, Sandra; Monti, Martina; Castagnini, Cinzia; Solito, Raffaella; Botta, Maurizio; Schenone, Silvia; Giachetti, Antonio; Ziche, Marina

    2007-03-01

    Src tyrosine kinase family cooperates with activated growth factor receptors to regulate growth, invasion and metastasis. The authors examined the influence of a novel c-Src inhibitor, 1l, derived from 4-amino-substituted-pyrazolo-pyrimidines, on tumor angiogenesis and on the angiogenic output of squamous carcinoma cells, A431 and SCC-4. The effect of 1l was assessed on growth and microvessel density in A431 tumors and its effect compared with the established c-Src inhibitor PP-1. The effects of c-Src inhibition were investigated on vascular endothelial growth factor (VEGF) expression and activity in tumor cells grown in vivo and in vitro, as well as on VEGF mediated signaling and on endothelial cell functions. Nanomolar concentrations of 1l decreased tumor volume promoted by A431 implanted in nude mice, without affecting in vitro cell tumor survival. This effect was related to 1l inhibition of VEGF production, and secondary to an effect on tumor microvessel density. The rabbit cornea assay confirmed that 1l markedly decreased neovessel growth induced by VEGF. In cultured endothelial cells, 1l inhibited the VEGF-induced phosphorylation on tyr416 of c-Src, resulting in a reduced cell proliferation and invasion. Consistently, 1l dowregulated endothelial nitric oxide synthase, MAPK-extracellular receptor kinase 1-2 (ERK1-2) activity and matrix metalloproteinases (MMP-2/MMP-9), while the tissue inhibitors of metalloproteinases (TIMP2/TIMP-1) were upregulated. These results demonstrate that nM concentrations of c-Src kinase inhibitors (1l and PP-1), by reducing the production of VEGF released by tumor cell and its endothelial cell responses, have a highly selective antiangiogenesis effect, which might be useful in combination therapies.

  7. Development of Nano-Liposomal Formulations of Epidermal Growth Factor Receptor Inhibitors and their Pharmacological Interactions on Drug-Sensitive and Drug-Resistant Cancer Cell Lines

    NASA Astrophysics Data System (ADS)

    Trummer, Brian J.

    A rapidly expanding understanding of molecular derangements in cancer cell function has led to the development of selective, targeted chemotherapeutic agents. Growth factor signal transduction networks are frequently activated in an aberrant fashion, particularly through the activity of receptor tyrosine kinases (RTK). This has spurred an intensive effort to develop receptor tyrosine kinase inhibitors (RTKI) that are targeted to specific receptors, or receptor subfamilies. Chapter 1 reviews the pharmacology, preclinical, and clinical aspects of RTKIs that target the epidermal growth factor receptor (EGFR). EGFR inhibitors demonstrate significant success at inhibiting phosphorylation-based signaling pathways that promote cancer cell proliferation. Additionally RTKIs have physicochemical and structural characteristics that enable them to function as inhibitors of multi-drug resistance transport proteins. Thus EGFR inhibitors and other RTKIs have both on-target and off-target activities that could be beneficial in cancer therapy. However, these agents exert a number of side effects, some of which arise from their hydrophobic nature and large in vivo volume of distribution. Side effects of the EGFR inhibitor gefitinib include skin rash, severe myelotoxicity when combined with certain chemotherapeutic agents, and impairment of the blood brain barrier to xenobiotics. Weighing the preclinical and clinical observations with the EGFR inhibitors, we developed the primary overall hypothesis of this research: that drug-carrier formulations of RTKIs such as the EGFR inhibitors could be developed based on nanoparticulate liposomal carriers. Theoretically, this carrier strategy would ameliorate toxicity and improve the biodistribution and tumor selectivity of these agents. We hypothesized specifically that liposomal formulations could shift the biodistribution of EGFR inhibitors such as gefitinib away from skin, bone marrow, and the blood brain barrier, and toward solid tumors

  8. Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum.

    PubMed

    Tripathi, Jaindra N; Lorenzen, Jim; Bahar, Ofir; Ronald, Pamela; Tripathi, Leena

    2014-08-01

    Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern-recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21-mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar 'Gonja manjaya' (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.

  9. Analysis of nucleotide diversity among alleles of the major bacterial blight resistance gene Xa27 in cultivars of rice (Oryza sativa) and its wild relatives.

    PubMed

    Bimolata, Waikhom; Kumar, Anirudh; Sundaram, Raman Meenakshi; Laha, Gouri Shankar; Qureshi, Insaf Ahmed; Reddy, Gajjala Ashok; Ghazi, Irfan Ahmad

    2013-08-01

    Xa27 is one of the important R-genes, effective against bacterial blight disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo). Using natural population of Oryza, we analyzed the sequence variation in the functionally important domains of Xa27 across the Oryza species. DNA sequences of Xa27 alleles from 27 rice accessions revealed higher nucleotide diversity among the reported R-genes of rice. Sequence polymorphism analysis revealed synonymous and non-synonymous mutations in addition to a number of InDels in non-coding regions of the gene. High sequence variation was observed in the promoter region including the 5'UTR with 'π' value 0.00916 and 'θ w ' = 0.01785. Comparative analysis of the identified Xa27 alleles with that of IRBB27 and IR24 indicated the operation of both positive selection (Ka/Ks > 1) and neutral selection (Ka/Ks ≈ 0). The genetic distances of alleles of the gene from Oryza nivara were nearer to IRBB27 as compared to IR24. We also found the presence of conserved and null UPT (upregulated by transcriptional activator) box in the isolated alleles. Considerable amino acid polymorphism was localized in the trans-membrane domain for which the functional significance is yet to be elucidated. However, the absence of functional UPT box in all the alleles except IRBB27 suggests the maintenance of single resistant allele throughout the natural population.

  10. mTOR inhibitors block Kaposi sarcoma growth by inhibiting essential autocrine growth factors and tumor angiogenesis.

    PubMed

    Roy, Debasmita; Sin, Sang-Hoon; Lucas, Amy; Venkataramanan, Raman; Wang, Ling; Eason, Anthony; Chavakula, Veenadhari; Hilton, Isaac B; Tamburro, Kristen M; Damania, Blossom; Dittmer, Dirk P

    2013-04-01

    Kaposi sarcoma originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi sarcoma-associated herpesvirus (KSHV) are endemic, Kaposi sarcoma is the most common cancer overall, but model systems for disease study are insufficient. Here, we report the development of a novel mouse model of Kaposi sarcoma, where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, and RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results show that mTOR inhibitors exert a direct anti-Kaposi sarcoma effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for Kaposi sarcoma and other cancers of endothelial origin.

  11. Impact damage resistance of carbon/epoxy composite tubes for the DC-XA liquid hydrogen feedline

    NASA Technical Reports Server (NTRS)

    Nettles, A. T.

    1995-01-01

    Low-velocity impacts were inflicted upon two elbow sections of carbon/epoxy feedline that are to be a part of the Delta Clipper-XA flight vehicle. A soap-based liquid leak detector solution was used to inspect the impact sites for leaks of pressurized gas that was pumped into the tube. Visual surface damage was noted and recorded for each impact site. After impact testing of each of the two sections of tubes was completed, the damage zones were disected from the tube and cross sectioned through the impact site. These specimens were polished after potting them in epoxy and were examined for microcracking using a fluorescent dye penetrant technique. The results showed that nonvisible damage could cause microcracking, thereby resulting in leaks through the tube wall.

  12. [Inhibitor development after early high exposure and cerebral haemorrhage. Costs and factor demand for a successful immunotolerance induction therapy].

    PubMed

    Haubold, K; Moorthi, C; Bade, A; Niekrens, C; Auerswald, G

    2010-11-01

    Severe haemophilia A was diagnosed postpartum in a newborn. The mother was known as a conductor (intron 22 inversion) and an uncle had a persistently high titer inhibitor after failed ITI. Due to a cephalhaematoma, a high-dose pdFVIII substitution was given within the first days after birth. At the age of six month a severe cerebral haemorrhage occurred, making a high-dose pdFVIII substitution and neurosurgical intervention necessary. Several days later a porth-a-cath-system was implanted. The development of a high titer inhibitor occured six days later, an ITI was started according to the Bonn Protocol. Initially rFVIIa was given in addition to the pdFVIII substitution. Seven days after the beginning of treatment the inhibitor was no longer detectable. At monthly intervals the FVIII dosage was reduced until the dosage complied with a prophylaxis in severe haemophilia A. The duration of the ITI was nine months. A total of 30 mg rFVIIa and 276000 IU pdFVIII were used; costs in total: 280173.60 Euro.

  13. Association between tumour necrosis factorinhibitors and risk of serious infections in people with inflammatory bowel disease: nationwide Danish cohort study

    PubMed Central

    Pasternak, Björn; Friis-Møller, Nina; Andersson, Mikael; Jess, Tine

    2015-01-01

    Objective To investigate whether people with inflammatory bowel disease treated with tumour necrosis factor-α (TNF-α) inhibitors are at increased risk of serious infections. Design Nationwide register based propensity score matched cohort study. Setting Denmark, 2002-12. Participants The background cohort eligible for matching comprised 52 392 people with inflammatory bowel disease, aged 15 to 75 years, of whom 4300 were treated with TNF-α inhibitors. To limit confounding, a two stage matching method was applied; firstly matching on age, sex, disease duration, and inflammatory bowel disease subtype, and secondly matching on propensity scores (1:1 ratio); this yielded 1543 people treated with TNF-α inhibitors and 1543 untreated to be included in the analyses. Main outcome measures The main outcome was any serious infection, defined as a diagnosis of infection associated with hospital admission. Cox regression was used to estimate hazard ratios for two risk periods (90 and 365 days after the start of TNF-α inhibitor treatment). Hazard ratios of site specific serious infections were obtained solely for the 365 days risk period. Results Within the 90 days risk period, 51 cases of infection were observed in users of TNF-α inhibitors (incidence rate 14/100 person years), compared with 33 cases in non-users (9/100 person years), yielding a hazard ratio of 1.63 (95% confidence interval 1.01 to 2.63). Within the risk period of 365 days, the hazard ratio was 1.27 (0.92 to 1.75). In analyses of site specific infections, the hazard ratio was above 2 for several of the subgroups but only reached statistical significance for skin and soft tissue infections (2.51, 1.23 to 5.12). Conclusions This nationwide propensity score matched cohort study suggests an increased risk of serious infections associated with use of TNF-α inhibitors within the first 90 days of starting treatment and a subsequent decline in risk. This calls for increased clinical awareness of potential

  14. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

    PubMed

    Robert, Carine; Nagaria, Pratik K; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J; Cole, Philip A; Rassool, Feyruz V

    2016-06-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells.

  15. A combination of 2D similarity search, pharmacophore, and molecular docking techniques for the identification of vascular endothelial growth factor receptor-2 inhibitors.

    PubMed

    Ai, Guanhua; Tian, Caiping; Deng, Dawei; Fida, Guissi; Chen, Haiyan; Ma, Yuxiang; Ding, Li; Gu, Yueqing

    2015-04-01

    The human vascular endothelial growth factor receptor-2 (VEGFR-2) has been an attractive target for the inhibition of angiogenesis. In the current study, we used a hybrid protocol of virtual screening methods to retrieve new VEGFR-2 inhibitors from the Zinc-Specs Database (441 574 compounds). The hybrid protocol included the initial screening of candidates by comparing the 2D similarity to five reported top active inhibitors of 13 VEGFR-2 X-ray crystallography structures, followed by the pharmacophore modeling of virtual screening on the basis of receptor-ligand interactions and further narrowing by LibDOCK to obtain the final hits. Two compounds (AN-919/41439526 and AK-968/40939851) with a high libscore were selected as the final hits for a subsequent cell cytotoxicity study. The two compounds screened exerted significant inhibitory effects on the proliferation of cancer cells (U87 and MCF-7). The results indicated that the hybrid procedure is an effective approach for screening specific receptor inhibitors.

  16. Design, synthesis and biological evaluation of novel 4-anilinoquinazolines with C-6 urea-linked side chains as inhibitors of the epidermal growth factor receptor.

    PubMed

    Zhang, Xu; Peng, Ting; Ji, Xun; Li, Jian; Tong, Linjiang; Li, Zeng; Yang, Wei; Xu, Yungen; Li, Mengyuan; Ding, Jian; Jiang, Hualiang; Xie, Hua; Liu, Hong

    2013-12-15

    A novel series of anilinoquinazoline compounds with C-6 urea-linked side chains was designed and synthesized as reversible inhibitors of epidermal growth factor receptor (EGFR) based on the structure-activity relationships (SARs) of anilinoquinazoline inhibitors. All compounds demonstrated good inhibition of EGFR wild type (EGFR wt) (IC50=0.024-1.715 μM) and inhibited proliferation of A431cell line (IC50=0.116-22.008 μM). The binding mode of compounds 8a, 8d, 8k and 8o was consistent with the biological results. Moreover, compounds 8k and 8l almost completely blocked the phosphorylation of EGFR in A431 cell line at 0.01 μM. Interestingly, all of the compounds also demonstrated moderate inhibition of EGFR/T790M/L858R (IC50=0.049-5.578 μM). In addition, compounds 8f and 8h blocked the autophosphorylation of EGFR in NCI-H1975 cells at high concentration (10 μM), and compound 8f was confirmed to be an irreversible inhibitor through the dilution method. Importantly, the compounds with C-6 urea-linked side chains which did not contain Michael acceptors demonstrated moderate to strong irreversible EGFR inhibition.

  17. Prevalence of IVS10nt-18G/A in Calabrian patients with moderate/mild hemophilia A and relation with Factor VIII inhibitor antibodies.

    PubMed

    Prejanò, Simona; Santoro, Rita C; Iannaccaro, Piergiorgio

    2015-10-01

    Hemophilia A is an X-linked bleeding disorder caused by widespread mutations in the factor VIII gene. In the course of a screening to research some hemophilia A mutations, our team has identified and posted a previously unreported nucleotide change in intron 10 in 20 patients with hemophilia A. We tried to identify a possible blood relationship between the people with this mutation, performing a backwards study of every family tree. First, we interviewed the patients and, if possible, parents and grandparents. When direct memory was no longer available, we consulted Registries of Births, Marriages and Deaths, and if these data were not sufficient, going backwards in time, we consulted registries of parish churches where newborns were baptized. The studied mutation was present in 33 hemophilic patients living in Calabria, 28 of them related. Three patients, carriers of this mutation, developed an FVIII inhibitor. In all the cases, the inhibitor development followed intensive treatments, after many days of exposure. Our study displayed the presence of a responsible moderate hemophilia A mutation, limited apparently to our country, probably because of a single ancestral event, and connected with FVIII inhibitor development.

  18. Programmed Application of Transforming Growth Factor β3 and Rac1 Inhibitor NSC23766 Committed Hyaline Cartilage Differentiation of Adipose-Derived Stem Cells for Osteochondral Defect Repair

    PubMed Central

    Zhu, Shouan; Chen, Pengfei; Wu, Yan; Xiong, Si; Sun, Heng; Xia, Qingqing; Shi, Libing

    2014-01-01

    Hyaline cartilage differentiation is always the challenge with application of stem cells for joint repair. Transforming growth factors (TGFs) and bone morphogenetic proteins can initiate cartilage differentiation but often lead to hypertrophy and calcification, related to abnormal Rac1 activity. In this study, we developed a strategy of programmed application of TGFβ3 and Rac1 inhibitor NSC23766 to commit the hyaline cartilage differentiation of adipose-derived stem cells (ADSCs) for joint cartilage repair. ADSCs were isolated and cultured in a micromass and pellet culture model to evaluate chondrogenic and hypertrophic differentiation. The function of Rac1 was investigated with constitutively active Rac1 mutant and dominant negative Rac1 mutant. The efficacy of ADSCs with programmed application of TGFβ3 and Rac1 inhibitor for cartilage repair was studied in a rat model of osteochondral defects. The results showed that TGFβ3 promoted ADSCs chondro-lineage differentiation and that NSC23766 prevented ADSC-derived chondrocytes from hypertrophy in vitro. The combination of ADSCs, TGFβ3, and NSC23766 promoted quality osteochondral defect repair in rats with much less chondrocytes hypertrophy and significantly higher International Cartilage Repair Society macroscopic and microscopic scores. The findings have illustrated that programmed application of TGFβ3 and Rac1 inhibitor NSC23766 can commit ADSCs to chondro-lineage differentiation and improve the efficacy of ADSCs for cartilage defect repair. These findings suggest a promising stem cell-based strategy for articular cartilage repair. PMID:25154784

  19. Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

    PubMed

    Maubach, Gunter; Sokolova, Olga; Wolfien, Markus; Rothkötter, Hermann-Josef; Naumann, Michael

    2013-09-15

    Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

  20. Statins suppress glucose-induced plasminogen activator inhibitor-1 expression by regulating RhoA and nuclear factor-κB activities in cardiac microvascular endothelial cells.

    PubMed

    Ni, Xiao-Qing; Zhu, Jian-Hua; Yao, Ning-Hua; Qian, Juan; Yang, Xiang-Jun

    2013-01-01

    The aim of this study was to investigate the possible proinflammatory signaling pathways involved in statin inhibition of glucose-induced plasminogen activator inhibitor-1 (PAI-1) expression in cardiac microvascular endothelial cells (CMECs). Primary rat CMECs were grown in the presence of 5.7 or 23 mmol/L glucose. PAI-1 mRNA and protein expression levels were measured by realtime polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay, respectively. A pull-down assay was performed to determine RhoA activity. IκBα protein expression was measured by Western blotting, nuclear factor (NF)-κB activation was detected by electrophoretic mobility shift assay and its transcription activity was determined by a dual luciferase reporter gene assay. PAI-1 mRNA and protein expression levels were both increased with high glucose concentrations, but they were significantly suppressed by simvastatin and atorvastatin treatment (P < 0.01) and the effects were reversed by mevalonate (100 μmol/L) and geranylgeranyl pyrophosphate (10 μmol/L) but not farnesyl pyrophosphate (10 μmol/L). Such effects were similar to those of a RhoA inhibitor, C3 exoenzyme (5 μg/mL), inhibitors of RhoA kinase (ROCK), Y-27632 (10 μmol/L) and hydroxyfasudil (10 μmol/L) and an NF-κB inhibitor, BAY 11-7082 (5 μmol/L). High glucose-induced RhoA and NF-κB activations in CMECs were both significantly inhibited by statins (P < 0.01). Simvastatin and atorvastatin equally suppress high glucose-induced PAI-1 expression. These effects of statins may occur partly by regulating the RhoA/ROCK-NF-κB pathway. The multifunctional roles of statins may be particularly beneficial for patients with metabolic syndrome.

  1. Drug efflux by Breast Cancer Resistance Protein (BCRP) is a mechanism of resistance to the benzimidazole insulin-like growth factor receptor/insulin receptor inhibitor, BMS-536924

    PubMed Central

    Hou, Xiaonan; Huang, Fei; Carboni, Joan M.; Flatten, Karen; Asmann, Yan W.; Eyck, Cynthia Ten; Nakanishi, Takeo; Tibodeau, Jennifer D.; Ross, Douglas D.; Gottardis, Marco M.; Erlichman, Charles; Kaufmann, Scott H.; Haluska, Paul

    2010-01-01

    Background Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types, including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors BMS-554417 and BMS-536924 and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared to MCF-7 cells, BCRP expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (p= 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared to MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807 did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ABC transporter BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism. PMID:21220496

  2. Rapid loss of factor XII and XI activity in ellagic acid-activated normal plasma: role of plasma inhibitors and implications for automated activated partial thromboplastin time recording.

    PubMed

    Joist, J H; Cowan, J F; Khan, M

    1977-12-01

    Rapid prolongation of the aPTT of normal plasma upon incubation with ellagic acid containing aPTT reagents was observed. The aPTT prolongation was not due to time-dependent changes in pH in the incubation mixture or loss of activity of the labile coagulation factors VIII and V but occurred as a result of rapid progressive inactivation of ellagic acid-activated factors XII and XI. Prolongation of the aPTT and loss of contact factor activities was not observed in plasma incubated with particulate activator reagents. This finding seemed to indicate that adsorption of factors XII and XI to larger particles during the activation process might protect these factors from inactivation by naturally occurring plasma inhibitors. Evidence is presented which supports previous findings that C1-INH, alpha1-AT, and antithrombin (in the presence of heparin) contribute to factor XIIa and XI a inactivation in ellagic acid-activated plasma and that plasma albumin may compete with factor XII for ellagic acid binding. The data indicate that ellagic acid-containing aPTT reagents have unfavorable properties which seriously limit their usefulness in the clinical laboratory, particularly in respect to recording of the aPTT with certain fully automated clot timers.

  3. Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

    PubMed Central

    Ménard, Robert; Schoenhofen, Ian C.; Tao, Limei; Aubry, Annie; Bouchard, Patrice; Reid, Christopher W.; Lachance, Paule; Twine, Susan M.; Fulton, Kelly M.; Cui, Qizhi; Hogues, Hervé; Purisima, Enrico O.

    2014-01-01

    Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 μM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 μM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 μM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella. PMID:25267679

  4. Discovery of Novel Insulin-Like Growth Factor-1 Receptor Inhibitors with Unique Time-Dependent Binding Kinetics

    PubMed Central

    2013-01-01

    This letter describes a series of small molecule inhibitors of IGF-1R with unique time-dependent binding kinetics and slow off-rates. Structure–activity and structure–kinetic relationships were elucidated and guided further optimizations within the series, culminating in compound 2. With an IGF-1R dissociative half-life (t1/2) of >100 h, compound 2 demonstrated significant and extended PD effects in conjunction with tumor growth inhibition in xenograft models at a remarkably low and intermittent dose, which correlated with the observed in vitro slow off-rate properties. PMID:24900721

  5. Susceptibility to natural killer cell-mediated lysis of colon cancer cells is enhanced by treatment with epidermal growth factor receptor inhibitors through UL16-binding protein-1 induction.

    PubMed

    Bae, Jae-Ho; Kim, So-Jung; Kim, Mi-Ju; Oh, Sae-Ock; Chung, Joo-Seop; Kim, Sun-Hee; Kang, Chi-Dug

    2012-01-01

    We have previously shown that inhibition of intracellular signaling pathways by treatment with quercetin induced the expression of natural killer cell group 2D (NKG2D) ligands on cancer cells and made the cells sensitive to natural killer (NK)-cell mediated cytotoxicity. In the present study, we investigated whether epidermal growth factor receptor (EGFR) inhibitors could induce the expression of NKG2D ligands in colon cancer cells. Treatment with EGFR inhibitors predominantly increased the levels of mRNA transcripts and surface protein of UL16-binding protein-1 (ULBP1) in various colon cancer cells, including KM12, Caco-2, HCT-15, and HT-29, which express EGFR, and increased susceptibility of these colon cancer cells to NK-92 cells. The expression of ULBP1 was not induced by inhibitors of nuclear factor-κB, phosphatidylinositol 3 kinase, and MAPK, but was induced by inhibitors of PKC, and the induction of ULBP1 expression with EGFR inhibitors was prevented by treatment with PMA in colon cancer cells. A transcription factor, activator protein-2 alpha (AP-2α), which has a suppressive effect on ULBP1 transcription, was prevented from binding to the ULBP1 promoter by treatment with EGFR inhibitors. The present study suggests that EGFR inhibitors can enhance the susceptibility to NK cell-mediated lysis of colon cancer cells by induction of ULBP1 via inhibition of the PKC pathway.

  6. Isolation and properties of a blood coagulation factor X activator from the venom of king cobra (Ophiophagus hannah).

    PubMed

    Lee, W H; Zhang, Y; Wang, W Y; Xiong, Y L; Gao, R

    1995-10-01

    A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

  7. Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

    PubMed Central

    Morkunas, Bernardas; Gal, Balint; Galloway, Warren R J D; Hodgkinson, James T; Ibbeson, Brett M; Sing Tan, Yaw; Welch, Martin

    2016-01-01

    Summary Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable. PMID:27559393

  8. Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes.

    PubMed

    Walter, Justin D; Werther, Rachel A; Brison, Caileen M; Cragerud, Rebecca K; Healey, John F; Meeks, Shannon L; Lollar, Pete; Spiegel, P Clint

    2013-12-19

    The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by 2 classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously was investigated by x-ray crystallography. The C2 domain/3E6 FAB/G99 FAB ternary complex illustrates that each antibody recognizes epitopes on opposing faces of the factor VIII C2 domain. The 3E6 epitope forms direct contacts to the C2 domain at 2 loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, whereas the G99 epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282, and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charge present on both C2 epitopes and complementary negative charge on each antibody. A new model of membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. These results illustrate the potential complexities of the polyclonal anti-factor VIII immune response and further define the "classical" and "nonclassical" types of antibody inhibitors against the factor VIII C2 domain.

  9. An integrated approach to ligand- and structure-based drug design: development and application to a series of serine protease inhibitors.

    PubMed

    Nicolotti, Orazio; Miscioscia, Teresa Fabiola; Carotti, Andrea; Leonetti, Francesco; Carotti, Angelo

    2008-06-01

    A novel approach was developed to rationally interface structure- and ligand-based drug design through the rescoring of docking poses and automated generation of molecular alignments for 3D quantitative structure-activity relationship investigations. The procedure was driven by a genetic algorithm optimizing the value of a novel fitness function, accounting simultaneously for best regressions among binding-energy docking scores and affinities and for minimal geometric deviations from properly established crystal-based binding geometry. The GRID/CPCA method, as implemented in GOLPE, was used to feature molecular determinants of ligand binding affinity for each molecular alignment. In addition, unlike standard procedures, a novel multipoint equation was adopted to predict the binding affinity of ligands in the prediction set. Selectivity was investigated through square plots reporting experimental versus recalculated binding affinities on the targets under examination. The application of our approach to the modeling of affinity data of a large series of 3-amidinophenylalanine inhibitors of thrombin, trypsin, and factor Xa generated easily interpretable and independent models with robust statistics. As a further validation study, our approach was successfully applied to a series of 3,4,7-substituted coumarins, acting as selective MAO-B inhibitors.

  10. 3D pharmacophore-based virtual screening, docking and density functional theory approach towards the discovery of novel human epidermal growth factor receptor-2 (HER2) inhibitors.

    PubMed

    Gogoi, Dhrubajyoti; Baruah, Vishwa Jyoti; Chaliha, Amrita Kashyap; Kakoti, Bibhuti Bhushan; Sarma, Diganta; Buragohain, Alak Kumar

    2016-12-21

    Human epidermal growth factor receptor 2 (HER2) is one of the four members of the epidermal growth factor receptor (EGFR) family and is expressed to facilitate cellular proliferation across various tissue types. Therapies targeting HER2, which is a transmembrane glycoprotein with tyrosine kinase activity, offer promising prospects especially in breast and gastric/gastroesophageal cancer patients. Persistence of both primary and acquired resistance to various routine drugs/antibodies is a disappointing outcome in the treatment of many HER2 positive cancer patients and is a challenge that requires formulation of new and improved strategies to overcome the same. Identification of novel HER2 inhibitors with improved therapeutics index was performed with a highly correlating (r=0.975) ligand-based pharmacophore model (Hypo1) in this study. Hypo1 was generated from a training set of 22 compounds with HER2 inhibitory activity and this well-validated hypothesis was subsequently used as a 3D query to screen compounds in a total of four databases of which two were natural product databases. Further, these compounds were analyzed for compliance with Veber's drug-likeness rule and optimum ADMET parameters. The selected compounds were then subjected to molecular docking and Density Functional Theory (DFT) analysis to discern their molecular interactions at the active site of HER2. The findings thus presented would be an important starting point towards the development of novel HER2 inhibitors using well-validated computational techniques.

  11. Cyclooxygenase-2 or tumor necrosis factorinhibitors attenuate the mechanotransductive effects of pulsed focused ultrasound to suppress mesenchymal stromal cell homing to healthy and dystrophic muscle.

    PubMed

    Tebebi, Pamela A; Burks, Scott R; Kim, Saejeong J; Williams, Rashida A; Nguyen, Ben A; Venkatesh, Priyanka; Frenkel, Victor; Frank, Joseph A

    2015-04-01

    Maximal homing of infused stem cells to diseased tissue is critical for regenerative medicine. Pulsed focused ultrasound (pFUS) is a clinically relevant platform to direct stem cell migration. Through mechanotransduction, pFUS establishes local gradients of cytokines, chemokines, trophic factors (CCTF) and cell adhesion molecules (CAM) in treated skeletal muscle that subsequently infused mesenchymal stromal cells (MSC) can capitalize to migrate into the parenchyma. Characterizing molecular responses to mechanical pFUS effects revealed tumor necrosis factor-alpha (TNFα) drives cyclooxygenase-2 (COX2) signaling to locally increase CCTF/CAM that are necessary for MSC homing. pFUS failed to increase chemoattractants and induce MSC homing to treated muscle in mice pretreated with ibuprofen (nonspecific COX inhibitor) or etanercept (TNFα inhibitor). pFUS-induced MSC homing was also suppressed in COX2-knockout mice, demonstrating ibuprofen blocked the mechanically induced CCTF/CAM by acting on COX2. Anti-inflammatory drugs, including ibuprofen, are administered to muscular dystrophy (MD) patients, and ibuprofen also suppressed pFUS-induced homing to muscle in a mouse model of MD. Drug interactions with cell therapies remain unexplored and are not controlled for during clinical cell therapy trials. This study highlights potentially negative drug-host interactions that suppress stem cell homing and could undermine cell-based approaches for regenerative medicine.

  12. Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex.

    PubMed

    Purohit, Vivek S; Ramani, Karthik; Sarkar, Rita; Kazazian, Haig H; Balasubramanian, Sathyamangalam V

    2005-05-06

    Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.